The majority of pathogenic microorganisms have developed resistance to commercial antibiotics. It causes the risk of illness relapse with current antimicrobial therapy regimens; additional and/or different antibacterial drugs are needed to treat diseases caused by these pathogenic microorganisms. The applied analysis in the present study was purification and characterization of plant peptides isolated from the leaves of Fagonia bruguieri as well as their antibacterial activities against Gram-positive bacteria, Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, and methicillin-resistant Staphylococcus aureus, in addition to Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa. The minimum inhibitory concentration for the isolated peptide ranges from 25 to 62.5 mg/mL. The methanolic solvent was used for the extraction followed by reversed-phase high-performance liquid chromatography for purification of peptides. Eventually, the peptide characterization and identification were also determined by MALDI-TOF/TOF and SEM analysis. This study paves a way to the effective antimicrobials from the plant resources.