Antibiotic resistance has significantly constricted the therapeutic efficacy of antibiotics on treating bacterial infections, creating an urgent need for new antimicrobial agents. However, progress in developing these agents is slow, prompting the exploration of innovative strategies such as combination therapy. This study examined the combined effects of Temporin antimicrobial peptides with antibiotics against Escherichia coli and Pseudomonas aeruginosa. We found that Temporin-GHaR (GHaR) and Temporin-GHaK (GHaK) with polymyxin B (PMB) showed synergistic antibacterial activity against P. aeruginosa and E. coli, with a fractional inhibitory concentration index (FICI) below 0.5. Further mechanistic studies revealed that the combination of GHaR/GHaK with PMB significantly enhanced the bactericidal effect by synergistically augmenting the disruption of the outer and inner membranes of P. aeruginosa and E. coli. Meanwhile, this combination regimen demonstrated efficacy in inhibiting the biofilm formation of P. aeruginosa and E. coli at concentrations lower than those required for single-drug treatments, and enhanced the disruption of mature E. coli biofilms. The synergistic use of GHaR/GHaK and PMB augmented the antibacterial efficacy against P. aeruginosa and E. coli without increasing hemolytic toxicity to murine erythrocytes. These findings provide an experimental foundation and theoretical framework for the application of conventional antimicrobial agents.