Cyclotides are antimicrobial peptides and play significant role as bio preservative in the food industry. Since foodborne diseases are a universal issue, in the present study the different methods for partial purification of cyclotide like peptides from Viola tricolor were compared and the antimicrobial activities of these peptides were investigated on Staphylococcus aureus Escherichia coli, which are important in foodborne diseases. Cyclotide like peptides from the aerial parts of the plant were extracted and it was partially purified with three methods including reverse phase chromatography, two phase system with reverse phase chromatography and ammonium sulfate precipitation. The samples were analyzed by SDS-PAGE, Tricine–PAGE, and reverse phase HPLC. It was found that the first method has lower MIC (Minimum Inhibitory Concentration) and is the best method.