The present study was undertaken to evaluate the prevalence, underlying resistance mechanism, and virulence involved in Pseudomonas aeruginosa (n = 35) isolated from freshwater fishes in Andhra Pradesh, India. Antibiogram studies revealed that 68.5, 62.8, 37.1, 11.4, 8.5, 57.1, 54.2, and 48.5% of isolates had resistance to oxytetracycline, co-trimoxazole, doxycycline, enrofloxacin, ciprofloxacin, cefotaxime, ceftazidime, and ampicillin, respectively. The resistant isolates harboured the tetA (85.7%), tetD (71.4%), tetM (91.4%), sul1 (80%), blaCTX-M (57.1%), blaTEM (42.8%), and blaSHV (48.5%) genes. In total, 50% of the isolates were altered as multi-drug resistant, and the multiple antibiotic resistance index was calculated as 0.4. Furthermore, 37.3, 48.5, and 14.2% of isolates were categorized as strong, moderate, and weak biofilm formers, possessing pslA (91.5%) and pslD (88.6%) biofilm encoding genes. In total, 82.8% of the isolates exhibited efflux pump activity and harboured the mexA (74.2%), mexB (77.1%), and oprM (37.1%) genes. Virulent genes oprL, toxA, exoS, and phzM were detected in 68.5, 68.5, 100, and 17.1% of isolates, respectively. The data suggested that P. aeruginosa harbours multiple resistance mechanisms and virulence factors that may contribute to antibiotic resistance and pathogenicity, and their distribution in fish culture facilities highlights the public health hazards of the food chain.