Wastes generated from hotspot environments contain many antibiotics and pathogens that play a substantial role in disseminating antibiotic-resistant bacteria into the environment. The antibiotic resistance profile of bacteria from hotspot environments is overlooked in developing countries, particularly Ethiopia. There is also a growing interest in discovering novel classes of antibiotics from microorganisms that have different mechanisms of action worldwide to overcome antibiotic resistance. Although public awareness of antibiotic resistance and use has been determined in different countries, information on the awareness of antibiotic resistance and use in the Amhara National Regional State is scarce. This study was conducted to identify bacteria in hotspot environments and determine their resistance profiles; isolate antibiotic-producing bacteria and evaluate their antagonistic activities against multidrug-resistant bacteria as well as to assess the knowledge, attitude, and practice of healthcare professionals, patients, and animal farm owners/workers toward antibiotic resistance and uses. A cross-sectional study was conducted to isolate bacteria in hotspot environments and determine their resistance profiles, isolate antibiotic-producing bacteria and evaluate their antagonistic activities against multidrug resistant bacteria isolated from hotspot environments. A survey was also conducted to assess the knowledge, attitude, and practice of healthcare professionals, patients, and animal farm owners/workers toward antibiotic resistance and uses from November 2019 to June 2021. A total of 126 waste and wastewater samples were aseptically collected, transported, and processed for bacteriological isolation, identification, and susceptibility testing following standard procedures. Antibiotic-producing bacteria were also isolated from waste and soil samples and tested against multidrug-resistant bacteria using the single line streak and agar well diffusion method. The minimum inhibitory and bactericidal concentrations of the extracts were determined using two-fold serial dilution and streak plate methods. Standard questionnaires were distributed to total of 460 participants to collect data on their knowledge, attitude, and practice on antibiotic resistance and use. Results revealed that a total of 411 bacterial isolates were recovered, with 122 (29.7%) bacterial isolates being isolated from medical wastewater. The highest prevalence of resistance was observed in isolates from beef waste and wastewater against tetracycline 12 (85.7%). Most bacterial isolates 96 (23.4%) were resistant to two antibiotics, and 35 (8.5%) and 20 (4.9%) isolates XIX showed possible extensive and pan-drug resistance. Among the isolated bacteria, 381 (92.7%) were resistant to at least one antibiotic, and the highest number of antibiotic resistances 71 (98.6%) was observed in Staphylococcus aureus. Two hundred and sixteen (52.6%) of the bacterial isolates showed multidrug resistance, and the highest multidrug resistance prevalence was observed in Pseudomonas spp. 47 (65.3%) isolates. The isolation source multi-antibiotic resistance index was greater than 0.2 in all hotspot environments, and the highest multi-antibiotic resistance index (0.47) was recorded from medical wastewater. The results showed that there was a statistically significant difference among hotspot environments for resistance to all antibiotics except ampicillin (p<0.05). The results also showed that antibiotic-producing bacterial isolates Ampb17 and Ampb33 obtained from soil and poultry waste showed the highest antagonistic activity against Salmonella spp., with a mean inhibition zone of 20.3±0.3 mm. The crude extract from isolate Ampb30 isolated from dairy waste showed the highest antagonistic activity against Salmonella spp., with a mean inhibition zone of 27.7±0.3 mm. One-way analysis of variance confirmed that most of the crude extracts against tested multidrug-resistant bacteria were statistically significant at a p < 0.05. Crude extracts of Ampb29, Ampb32, Ampb30, and Ampb33 showed minimum inhibitory concentrations of 0.13 mg/ml, and extracts from Ampb29, Ampb32, Ampb30, and Ampb5 showed a minimum bactericidal concentration of 0.25 mg/ml. Moreover, using the mean score as the cut-off point, most health care professionals 76 (55.9%) had good knowledge of antibiotic resistance and use. One hundred five (45.3%) patients and 48 (52.5%) animal farm owners/ workers had positive attitudes of antibiotic resistance and use. About 20 (88.2%) of healthcare professionals, 133 (57.3%) of patients, and 70 (76.9%) of animal farm owners/ workers indicated that poor patient adherence, poor handwashing practices, and poor awareness of antimicrobial resistance were significant factors for increasing antibiotic resistance, respectively. Moreover, 124 (91.2%) of healthcare professionals, 139 (59.9%) of patients, and 71 (78%) of animal farm owners/workers reported that establishing rapid and effective diagnostic techniques, implementing hygiene, infection prevention, and control practices, and increasing the use of complementary treatments were the most important interventions to reduce antibiotic resistance. It was concluded that wastes generated from hotspot environments and released into the environment contain large numbers of XX antibiotic-resistant, multidrug-resistant, possible extensively, and/or pan-drug-resistant bacteria. The results also revealed that antibiotic-producing bacteria from waste and soil are potential sources of antibacterial compounds. Moreover, the finding revealed that there are crucial gaps in the attitudes and practices of healthcare professionals, knowledge, attitudes, and practices of patients, and practice of animal farm owners/workers on antibiotic resistance and use. Proper waste management strategies, raising awareness, and implementing interventions are essential to reducing antibiotic resistance.