OBJECTIVE Bacteriophages are infectious replicating entities that are under consideration as antimicrobial bioagents to control bacterial infections. As an alternative or supplement to antibiotics, bacteriophages can be used to circumvent the resistance to existing antibiotics. The aim of this study was to assess the synergistic effect of a naturally isolated phage and ampicillin against Escherichia coli O157. METHODS In the present study, a natural phage against E. coli O157 was isolated, the morphology and molecular characteristics of the phage were identified, and the combination of bacteriophage and antibiotic to combat clinically isolated drug-resistant E. coli O157 was evaluated. RESULTS The results showed the synergistic action between a naturally isolated phage and ampicillin in solid (disk diffusion test) and liquid culture media. Addition of the isolated phage, gT0E.co-MGY2, to the microbial lawn of bacteria in modified antibiotic disk diffusion test, altered susceptibility pattern of E. coli O157 from resistant to sensitive based on the inhibition zones. Combinations of bacteriophage and ampicillin significantly enhanced the killing of bacterial strains when compared to treatment with ampicillin or phage alone in liquid culture. Moreover, it lasted few hours for ampicillin to reverse the growth of E. coli O157, while the bacteriophage and combination treatment stopped the proliferation of bacteria from the beginning, and this can compensate the delayed onset of antibiotic action. CONCLUSIONS The synergistic action of bacteriophages and antibiotics is an alternative that cannot only be effective against bacterial infections but also contribute to the reduction of antibiotic resistance.