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Mushrooms are considered one of the richest sources of natural antibiotics, and various species of them inhibit the growth of a wide diversity of microorganisms. Ganoderma lucidum, a well-known medicinal mushroom. has many pharmacological and biological activities including an antimicrobial effect, although few studies have investigated the antibacterial and antifungal effects of its purified compounds. The chemical structure of the purified compounds from the hexane fraction was elucidated as ergosta-7, 22-dien-3β-yl acetate, ergosta-5, 7, 22-trien-3β-yl acetate (isopyrocalciferol acetate), ergosta-7, 22-dien-3-one, ergosta-7, 22-dien-3β-ol, and ergosta-5, 7, 22-trien-3β-ol (ergostrol). In addition, the structure of ganodermadiol was demonstrated after purification from the chloroform fraction. The fractions inhibited Gram-positive bacteria and yeast, with minimum inhibitory concentration values of 6.25 mg/mL, but …