This study was aimed at the isolation and molecular characterization of bacteria associated with catfish from culture and wild environments. Samples of gills of Arius heudelotii and Claris gariepinus fishes of economic value from the New Calabar River Choba and earthen ponds from Aluu in Rivers State were collected and analysed. Characterization of isolates from the gills of the catfish was done using the 16S rRNA gene sequencing method to identify their relationship. The presence of a resistance gene was examined by polymerase chain reaction followed by agarose gel electrophoresis of the amplification products. The mean total heterotrophic bacterial counts was 6.5995±0.947 LogCFU/g for the gills’ samples obtained from the earthen pond Aluu. New Calabar River had 4.5041±1.054 LogCFU/g for the gills’ samples. A total of 12 bacterial flora were isolated from the gills of the sampled catfish: Escherichia coli, Klebsiella spp., Enterobacter spp., Citrobacter spp., Bacillus subtilis, Proteus vulgaris, Streptococcus spp., Shigella spp. Staphylococcus spp., Micrococcus spp., Vibrio spp. and Acinetobacter spp. Vibrio azureus, Aeromonas dhakensis, Proteus vulgaris, Aeromonas hydrophila. Vibrio azureus and Providencia alcalifaciens were found to be carriers of the TEM gene, while Providencia alcalifaciens carried the CTX-M gene. SHV gene was found in Vibrio azureus, Providencia alcalifacians, Proteus vulgaris, Aeromonas hydrophila, Providencia alcalifacians. However, the Oxa gene was not identified in the bacterial genera. The high probability of transferring them to other microbes to cause antibiotic pollution has become a serious public health challenge. Microbial loads with high or low presence of some potential pathogens call for public concern.