Abstract Antibiotic resistance represents a critical global health challenge driven by the dissemination of resistant bacterial genes across households, livestock and environmental reservoirs. Horizontal gene transfer and mutations play substantial roles in the existence and persistence of antimicrobial resistance significantly diminishing the effectiveness of current antibiotic therapies. This study aimed to investigate the genotypic and phenotypic resistance profiles of bacterial strains isolated from industrial effluent samples. Conducted in Nairobi County, Kenya, between January and December 2024, the study involved analysis of four bacterial strains isolated from industrial effluent samples collected via systematic sampling across multiple industrial sites. Bacterial identification was performed using API® 20E biochemical identification kit (BioMérieux, France). Antibiotic susceptibility testing encompassed several antibiotic classes including cephalosporins, penicillins, aminoglycosides, sulfonamides, tetracyclines, fluoroquinolones and carbapenems. DNA extraction from antibiotic-resistant isolates was conducted using ZyppyTM Plasmid Miniprep Kit (Zymo Research, USA) following the manufacturer’s protocol. Conventional PCR assays targeted resistance genes bla-TEM, bla-OXA, bla-KPC-1, bla-NDM and ParC. Sequence alignment was performed using MUSCLE software while phylogenetic analyses were conducted with MEGA 11 employing the Maximum Likelihood method to infer evolutionary relationships. All tested bacterial isolates including Pseudomonas aeruginosa, Escherichia coli, Aeromonas spp., Klebsiella pneumoniae, and Bacillus spp., exhibited 100% resistance to penicillin.  Resistance to tetracyclines, cephalosporins and sulfonamides was notably prevalent in Aeromonas spp. In contrast, carbapenems and aminoglycosides maintained substantial efficacy particularly against Aeromonas spp. and Pseudomonas aeruginosa strains. Multiplex PCR analysis revealed widespread distribution of resistance genes with bla-TEM being the most prevalent followed by bla-KPC, bla-OXA, bla-NDM and ParC underscoring the molecular basis for the observed resistance phenotypes. Phylogenetic analysis demonstrated high sequence homology with globally distributed pathogenic strains highlighting the clinical relevance and potential public health impact of these findings. These findings underscore the urgent need to integrate comprehensive surveillance systems and implement multifaceted antimicrobial strategies to curb the spread of multidrug-resistant bacterial strains across environmental, healthcare, and aquatic ecosystems.