A Cross-sectional study was conducted to assess the hygienic practices of fish handlers and to evaluate bacterial load and antimicrobial resistance patterns of E. coli isolates from fish value chain in Bahir Dar city from November 2019 to May 2020. From a total of 180 fish samples collected by systematic (for raw fish sampling) and purposive sampling techniques(for cooked fish sampling), 20% (36/180) of fish samples were positive for E.col in the three species of fish (Nile tilapia, African catfish and Labeo Barbus). From 115 uncooked and 65 cooked fish samples examined, 23.5 %( 27/115) and 13.8 %( 9/65) had E. coli, which was not statistically significant (p=0.10, OR=2.06, CI= 0.86-4.94). The bacterial load of uncooked and cooked fish samples were assessed by the aerobic plate count. The highest mean bacterial count was observed in raw fish samples (6.13×105 cfu/g), than cooked fish samples (2.81×104cfu/g). Among the interviewed fish handlers, 83.3%, 76.7% and 80% of respondents had good knowledge and attitude on using of clean cutting and filleting board, storing of raw and cooked foods separately and using of apron for reducing of risk of fish contamination, respectively. E.coli isolates were tested with seven available antimicrobial drugs. All 36 isolates were 100% sensitive to ciprofloxacin and gentamycin. E.coli isolates subjected to tetracycline were 55.6%, resistant, 8.3% intermediate and 36.1% susceptible. For trimethoprim-sulfamethoxazole, 25% were resistant and 75% were susceptible. This study revealed that there was a gap on food safety practices and high E.coli profile were observed according to the bench marks of center for food safety standards. Hence, it could be wise to advice the fish harvesters, fish traders, hotels and restaurants from harvesting to consumption to improve the food safety practices and quality standards of fish harvested and sold in the city. Keywords: Bahir Dar; E.coli; Fish; Hygienic Practice; Lake Tana