Background: The efflux pumps are one of the main mechanisms of the antibiotic resistance in Escherichia coli .The efflux pump inhibitors (chlorpromazine and omeprazole) were tested for their effect on the antibiotic resistance by inhibiting efflux pump activity. Objective: The present study aims to estimate the effect of some efflux pump inhibitors on the antibiotic resistance of some Escherichia coli isolates. Methodology: A total of 100 isolates of Escherichia coli were studied for antibacterial susceptibility pattern by disk diffusion method with and without efflux pump inhibitors chlorpromazine (25 µg) and omeprazole (100 µg), determination of the MIC of amikacin and gentamicin on 60 E.coli resistant isolates, the effect of the efflux pump inhibitors on the MIC of amikacin and gentamicin and PCR amplification of the efflux pump genes AcrD and MdfA genes. Results: The difference between all tested antibiotics in the change of resistance to totally sensitive E.coli isolates after addition of CPZ and OMP by disk diffustion method were statistically highly significant (p value <0.001), in which the highest percentage value were reported for aminoglycoside antibiotics (amikacin and gentamicin). The highest reduction in the MIC of amikacin and gentamicin was observed with chlorpromazine than omeprazole (p<0.05) .The proportion of isolates with greater than two-fold reductions in MIC in the presence of CPZ were 69.2% and 50.9% for amikacin and gentamicin respectively (p>0.05) while in the presence of OMP were 46.2% and 30.9% for amikacin and gentamicin respectively, (p>0.05). PCR detection of efflux pump genes detected a high level of AcrD gene detection than MdfA gene (p value <0.05).The percentage of AcrD detection in amikacin and gentamicin resistant isolates were 77% and 87.3% respectively, while for MdfA gene detection in amikacin and gentamicin resistant isolates were 59% and 71% respectively. Conclusion: Antibacterial efflux pumps are involved in establishment of resistance among the tested isolates. Chlorpromazine and Omperazole were capable of inhibiting the efflux pump with higher activity on aminoglycoside antibiotics. Chlorpromazine was more effective than Omeprazole as EPI. PCR results showed that AcrD and MdfA efflux pump genes contributed to the resistance of the tested aminoglycosides