OBJECTIVE Arginine-rich peptides are an important class of antimicrobial peptides (AMPs) that exert their antibacterial activity via a lytic mechanism. Although the antibacterial activity of arginine-rich peptides has been already evaluated, no reports have so far been evaluated the influence of reaction conditions on their antimicrobial potential. The aim of the present study was to investigate the influence of pH, temperature, and glycine on antibacterial activity of poly-l-arginine (PLA) with a molecular weight of 5-15 kDa against Escherichia coli O157:H7 and Staphylococcus aureus. METHODS The percentage of growth inhibition of PLA against both bacteria was analyzed at various pH, temperatures and sub-inhibitory concentrations of glycine by two-fold broth microdilution method. RESULTS The results showed that PLA had antibacterial activity against E. coli O157:H7 and S. aureus and the inhibitory effect increased with increasing PLA concentration. The antimicrobial activity of PLA against both microorganisms was higher in basic media than under acidic or neutral conditions. At 1/2 times the MIC, heat treatment intensified the toxicity of PLA against E. coli O157:H7 whereas the susceptibility to PLA seems to be temperature independent for S. aureus. The MICs of glycine against E. coli O157:H7 and S. aureus were 12.5 and 25 mg ml-1, respectively. The antibacterial activity of PLA against both microorganisms increased, as indicated by the increasing growth inhibition percentage of this peptide with increasing glycine concentration. CONCLUSIONS The antibacterial activity of PLA against S. aureus and E. coli O157:H7 depends on pH and glycine concentration.