Background:  Plasmid mediated Multi-Drug Resistant (MDR) Sul1 and ESBL resistance encoding genes are the major threats due to their ability to be transferred horizontally in any environment. In this study, the prevalence of Sul1 gene and ESBL coding genes (blaCTX-M, blaTEM or blaSHV) in E. coli and P. aeruginosa and determined their ability to transfer resistance through transformation.  Methods: Ninety-two trimethoprim-sulfamethoxazole resistant TSR isolates belonging to E. coli and P. aeruginosa species were obtained from clinical samples such as urine, wound, and blood from patients in 3 government hospitals in Delta State, Nigeria. Resistance to antimicrobial agents was determined by disc diffusion methods. PCR amplification was performed on extracted plasmid DNAs for the detection of ESBL and Sul1 genes using specific primers. Extracted plasmid DNAs of ESBL producing and Sul1 positive isolates were used in transforming a competent E. coli K-12   adopting CaCl₂ method. Results: All 92 TSR isolates were MDR with a vast majority of resistant patterns associated with the cephalosporins, amoxicillin-clavulanic acid, and the fluoroquinolones. Sul1 gene and ESBL genotypes were produced in 29.3% of E. coli and 58.7% of P. aeruginosa isolates, respectively. E. coli was more prevalent ESBL producer (75.9%) than P. aeruginosa (24.1%) and the blaCTX-M was the most prevalent gene (30.4%). Conclusion: Twenty-three isolates transferred several antibiotic resistances which includes ceftazidime, cefotaxime, cefuroxime, cefixime, gentamicin, ciprofloxacin, ofloxacin trimethoprim-sulfamethoxazole and amoxicillin-clavulanic acid, Sul1 gene and ESBL genotypes by transformation, thereby indicating a high potential for dissemination of resistance markers in hospitals. These findings are of health concern because of the rise in antimicrobial resistance associated with ESBL isolates.