This study evaluated the microbiological safety of fresh Nile tilapia (Oreochromis niloticus) from Kenyan fresh water fish value chains. One hundred seventy-six fish samples were analyzed. The microbial counts of hygiene indicators, total viable aerobic count (TVC), total coliforms, and fecal coliforms isolated by using culture techniques were enumerated, and microbial pathogens present in the fish samples were identified and characterized by using molecular methods. The diversity of bacterial isolates was determined by using the Shannon-Weaver diversity index. The mean of TVC in the samples was 4.44 log CFU/g. A comparison with the European Commission and International Commission on Microbiological Specifications for Foods standards showed two fish samples had counts above the 5.00 log CFU/g limit for TVC, and all the fish samples had total coliform and fecal coliform counts above 2.00 and 1.00 log CFU/g, respectively. Pathogenic strains, including Shiga toxin–producing and enteropathogenic Escherichia coli, Listeria monocytogenes, Yersinia enterocolitica, Klebsiella pneumoniae, and Salmonella enterica, were identified in the fish samples. The diversity of 1,608 bacterial isolates was higher in semiregulated chains than unregulated chains. The diversity was also high at the retail stage of the fish value chain. In conclusion, fresh Nile tilapia samples were above some of the set food safety standards and may be a source of foodborne pathogens. Further microbial risk assessment for detected pathogens is recommended to further support public health protection, taking into account growth, inactivation through cooking, processing, survival, and consumption.