BACKGROUND: Infections caused by drug-resistant strains of Acinetobacter baumannii and Pseudomonas aeruginosa are now a global problem that requires the immediate development of new antimicrobial drugs. Combination therapy and using antimicrobial peptides are two strategies with high potential to solve this issue. By these strategies, this study aimed to determine the antimicrobial effect of Nisin and P10 antimicrobial peptides on extensively drug-resistant Acinetobacter baumannii and colistin-resistant Pseudomonas aeruginosa isolates, and investigate the most effective combination of an antimicrobial peptide with an antibiotic. MATERIAL AND METHODS: This study was performed on five resistant clinical isolates and one standard strain for each kind of bacterium. First, the minimum inhibitory concentrations of two antimicrobial peptides (Nisin and P10) and five common antibiotics for the treatment of Gram-negative bacteria (ceftazidime, tobramycin, ciprofloxacin, doripenem, and colistin) was determined using Scanner-Assisted Colorimetric MIC Method. Then, the combination effect of P10+Nisin, P10+antibiotics, Nisin + antibiotics was investigated using checkerboard method. RESULTS: The MIC value of Nisin and P10 against studied pathogens were 64-256 and 8-32 μg/ml, respectively. P10+Nisin combination showed synergistic effect against standard strains and additive effect against drug-resistant clinical isolates. It was also found that the combination effect of P10+ceftazidim, P10+doripenem, and Nisin + colistin was synergistic in most cases. Nisin + tobramycin combination showed synergistic effect in exposure to standard strains, while the synergy is strain-dependent against drug-resistant clinical isolates. CONCLUSION: In conclusion, the synergism of Nisin + colistin and P10+ceftazidime/doripenem could be of great therapeutic value as antimicrobial drugs against infections caused by colistin-resistant P.aeruginosa and XDR A. baumannii.