An agar plate method was established to screen synergistic antibacterial agents other than beta-lactamase inhibitors. By using this method, a strain Aspergillus sp136 was selected for further studies. From the metabolites of this strain, a synergistic antibacterial compound was isolated by bioautographic TLC assay-guided fractionation and identified as helvolic acid. The synergistic effect of helvolic acid to penicillin was about 3 times that of clavulanic acid to penicillin in agar diffusion assay on Bacillus cereus. In checkerboard studies, helvolic acid exhibited synergistic effects with erythromycin on all tested multi-drug resistant Staphylococcus aureus and with penicillin and tetracycline on some multi-drug resistant S. aureus. A pattern of enhanced killing was also found in time-kill studies on multi-drug resistant S. aureus.