Extraction, isolation, and purification of an antimicrobial peptide was carried out from the flowers of Catharanthus roseus (white flower) and (pink flower) varieties and their antimicrobial activity was monitored against bacteria Escherichia coli (Gram-negative)and Staphylococcus aureus (Gram-positive). Cathranthus roseus is well known for its medicinal properties. This study illustrates the antibacterial property of this plant against pathogenic and opportunistic organisms like Escherichia coli and Staphylococcus aureus. The results showed that the Catharanthus roseus flowers (white) and (pink) purified extracts have antibacterial activity against Escherichia coli and Staphylococcus aureus. Therefore providing protection to the flowers against pathogen invasion. This study also shows that an antibacterial compound has been isolated and detected.