Peganum harmala is one of the most famous medicinal plants and natural products commonly used in traditional medicine and extensively spread in Middle and East Asia and North Africa. This study aimed to evaluate the radical scavenging activity of ethanolic crude extracts of P. harmala seeds from Libya using DPPH assay; to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) of the P. harmala seeds crude extracts against Gram-negative Salmonella typhi ATCC14028 and Escherichia coli ATCC 25922 and also Gram-positive Bacillus subtilis ATCC 6633 and Staphylococcus aureus ATCC 33591; and to screen the phytochemicals in the ethanolic crude extracts of the Libyan P. harmala seeds using gas chromatography-mass spectrometry (GC-MS). The IC50 value of P. harmala seeds extract was 179.62±7.32 μg/mL. The MIC ranged from 1.95 to 31.25 mg/mL while the MBC ranged from 7.80 to 62.50 mg/mL. The eight compounds identified were harmine, harmaline, leptalorine, hexahydro fluorene, 3-methoxy-6-methyl, 1H-pyrido [3,4-b]indole,2,34,9- tetrahydro-6methoxy-1-methyl, 9,12-octadienoic acid ethyl ester, linoleic acid ethyl ester, 9,12-octadienoic acid (z,z). This study has, therefore, revealed the antixodant and antibacterial efficacy of P. harmala ethanolic extract. Thus, it could be further developed as a substitute for chemical antioxidants and as antibacterials agent.