Fecal contaminants are a major public concern that directly affect human health in the fish production industry. In this study, we aimed to determine the fecal coliform, spoilage bacteria, and antimicrobial-resistant bacterial contamination in rainbow trout (Oncorhynchus mykiss) farms. Fish were sampled from rainbow trout farms that have a high production capacity and are established on spring water, stream water, and dammed lakes in six different regions of Turkey. A total of seven Enterobacter subspecies, two strains of Pseudomonas spp., and one isolate each of Morganella and Stenotrophomonas were characterized based on biochemical and molecular methods, including the 16S rRNA and gyrB housekeeping gene regions. The sequencing results obtained from the 16S rRNA and gyrB gene regions were deposited in the GenBank database and compared with isolates from different countries, which were registered in the database. Resistance to 10 different antimicrobial compounds was determined using the broth microdilution method, and molecular resistance genes against florfenicol, tetracycline, and sulfamethoxazole were identified by PCR. All detected resistance genes were confirmed by sequencing analyses. E. cloacae, E. asburiae, Pseudomonas spp., S. maltophilia, and M. psychrotolerans were identified using the gyrB housekeeping gene, while isolates showed different biochemical characteristics. All isolates were found to be phenotypically resistant to sulfamethoxazole, and some isolates were resistant to tetracycline, florfenicol, amoxicillin, and doxycycline; the resistance genes of these isolates included floR, tetC, tetD, and tetE. We showed that fecal coliforms, spoilage bacteria, and antimicrobial resistant bacteria were present in farmed rainbow trout, and they pose a threat for human health and must be controlled in the farming stage of fish production.