1-Octen-3-ol, known as mushroom alcohol, is a natural product extracted from fungi and plants. Its antimicrobial activities against five common food-related bacteria and two pathogenic fungi were evaluated in this paper, including Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Fusarium tricinctum and Fusarium oxysporum. The results showed that 1-octen-3-ol had a strong antibacterial activity against the tested bacteria, especially against Gram-positive bacteria, and it can also inhibit fungal growth and spore germination. The minimum inhibitory concentrations (MICs) for Gram-positive bacteria and Gram-negative bacteria were 1.0 and 2.0 mg/mL, respectively. The minimum bactericidal concentrations (MBCs) for Gram-positive bacteria and Gram-negative bacteria were 4.0 and 8.0 mg/mL, respectively. The completely inhibitory concentrations for fungal growth and spore germination were 8.0 and 2.0 mg/ml, respectively. Cell constituents’ leakage and scanning electron microscope assays indicated that 1-octen-3-ol changed the permeability of the cell membrane. Discrepant antimicrobial activity between 1-octen-3-ol and 1-octen-3-one indicated that hydroxyl may play a decisive role in antimicrobial activity. It is suggested that 1-octen-3-ol, with attractive mushroom aroma and antimicrobial activity, has potential applications in control of pathogens.