In this study, it was aimed to determine fatty acid profile and the antimicrobial activity of the seed oil obtained from the seed of Silybum marianum L. and silymarin which was extracted through methanol extraction of Silybum marianum L. seed. The antimicrobial activity of extracts was determined by testing against the microorganisms of Enterococcus faecalis, Staphylococcus aureus, Bacillus subtilis, Lactobacillus plantarum, Lactobacillus pentosus, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, and Candida albicans. Gas Chromatography-Mass Spectrometry was used to determine the fatty acid profile of the seed oil. The main components of the seed oil were detected as octadecadienoic acid (44.06 %), octadecenoic acid (20.18 %), hexadecenoic acid (14.63 %) and octadecanoic acid (7.75 %). Seed oil revealed antimicrobial effect on P. mirabilis, P. aeruginosa and L. pentosus ELB41 and silymarin had antimicrobial effect on L. plantarum ELB75, L. pentosus ELB37 and B. subtilis.