A cross-sectional study was carried out from October 2022 to June 2023 at Chagni cattle breeding and improvement Ranch, and small holder-dairy farms to estimate the prevalence and the anti-microbial susceptibility profile of Staphylococcus aureus isolated from infected cows. The study was conducted on 318 lactating cows selected by simple random sampling techniques in the study areas. The data was analysed using statistical tools. Based on the CMT result and clinical examination, of the 318 cows examined, 44.1%(140/318) were positive for mastitis. Out of this, 35.9% and 8.2% had sub-clinical and clinical mastitis at cow level, respectively. From 1272 quarters examined 55(4.3%) were blind, and 20% (243/1272) were positive for mastitis. In the multivariable logistic regression model, factors significantly associated (p < 0.05) with the occurrence of mastitis were age, breed, management system, lactation stage, parity, floor type, and tick on the teat at Chagni cattle breeding and improvement Ranch. In addition, age, lactation stage, and tick on teat were independently associated with mastitis occurrenc in small-holder dairy farms (p<0.05). Staphylococcus aureus isolates were detected in 45.7% (64/140) of the samples, of which 9 (34.6%) and 55 (48.2%) isolates from clinical and subclinical mastitis cases, respectively. All (n = 64) isolates of S. aureus were tested for their susceptibility to eight selected antimicrobials. The isolates were highly susceptible to sulfamethoxazole (87.5%) and Gentamycin (79.7 %) followed by Tetracycline (75%), Erythromycin (72%), and Azithromycin (71.8%). However, they were highly resistant to Cefoxitin (65.6%), followed by Tetracycline and ciprofloxacin (25%). The high prevalence of masitis in the study area, more importantly the sub-clinical one. And isolates of S. aureus were resistant to a number of drugs. Hence, implementing hygienic conditions, creating awareness on the control and prevention of subclinical mastitis in dairy farms, and rational use of drugs are recommended.