Disease outbreaks cause enormous financial losses and pose significant challenges to the aquaculture industry globally. This may lead to increased mortality rates, slower growth and productivity, greater production costs, and fewer revenue streams. The purpose of this study was to ascertain the prevalence and pattern of antibiotic susceptibility of Enterobacter cloacae isolated from diseased African catfish (Clarias gariepinus) in Lagos State, Nigeria. Aquaculture farms in three local government districts in Lagos State were the subject of this cross-sectional study. In Lagos State, 13 commercial farms located in three local governments (Surulere, Ikorodu, and Alimosho) provided a purposeful sample of 120 diseased African catfish (Clarias gariepinus). The 16S rRNA gene characterisation, biochemical testing, and morphological traits were used to identify the isolates. Using the agar disc diffusion method, the antimicrobial susceptibility pattern of Enterobacter cloacae was investigated. In infected Clarias gariepinus from Lagos State, the incidence of Enterobacter cloacae isolates was 60% (12/20) in Surulere, 76.7% (46/60) in Ikorodu, and 65% (26/40) in Alimosho. When comparing Ikorodu to other local governments, a noticeably greater prevalence was noted. Malaysian, Vietnamese, and Pakistani isolates of Enterobacter cloacae were closely related to the strains of OP595768.1 and OP595746.1 that were isolated. With a MAR value of 0.72, the isolates of Enterobacter cloacae were multidrug resistant. A study conducted in Lagos State revealed that Enterobacter cloacae, which was isolated from infected Clarias gariepinus, had evolutionary ancestry with harmful NCBI reference strains from various nations.