The use of plant extracts and their active compounds for medicinal purposes, particularly for antimicrobial activity, has become increasingly prevalent in recent years. This study aimed to evaluate the antimicrobial activities of Peganum harmala seed extract against various microorganisms using the disc diffusion method. Ethanol (65% concentration) was used to extract the active components from the P. harmala plant. Eight types of flavonoids in the plant extract were analyzed through HPLC. The antimicrobial activity of the extract was assessed against 15 microorganisms, including 14 bacterial strains and 1 fungus, via disc diffusion, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) tests. The results indicated that P. harmala exhibited activity against Bacillus subtilis, Candida albicans, Enterobacter aerogenes, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas fluorescens, Salmonella enteritidis, Salmonella infantis, Salmonella kentucky, Salmonella typhimurium, Staphylococcus aureus, and Staphylococcus epidermidis, with MIC values ranging from 6.50 µg/mL to 52.03 µg/mL. The P. harmala extract demonstrated both antibacterial and antifungal activity against all tested microorganisms, with varying efficacy.