Introduction: Oyster mushrooms are edible mushrooms that belong to the Pleurotaceae family. They contain bioactive compounds with numerous nutritional and health benefits. Studies of their antimicrobial activities have highlighted their natural role in biological control. Methods: Fresh oyster mushroom samples of Pleurotus ostreatus, Pleurotus florida, and Pleurotus sajor caju were collected from growers in Gujarat, India. They were sun-dried for four days and powdered. Ten grams of powder from each species were extracted with 300 ml of double-distilled water, methanol, and ethanol (70%) using a Soxhlet apparatus, depending on the boiling point of each solvent. Antimicrobial activity was evaluated by agar well diffusion against Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Candida parapsilosis using extracts prepared at a concentration of 100 mg/ml, and tested in volumes of 25, 50, and 100 µl. Results: Methanol and ethanol extracts of oyster mushrooms exhibited inhibition zones ranging from 3.9 to 4.8 mm, whereas the aqueous (double-distilled water) extracts showed comparatively lower activity. Alcoholic extracts produced the largest overall bacterial inhibition zones, ranging from 22 to 23 mm at the highest concentration. Among the tested bacteria, Staphylococcus aureus and Pseudomonas aeruginosa were the most sensitive. In contrast, the fungal strains showed smaller inhibition zones, with Candida albicans and Candida parapsilosis exhibiting zones of 12-13 mm. Among the tested species, the alcoholic extract of Pleurotus florida demonstrated slightly higher antibacterial activity than those of P. ostreatus and P. sajor caju. In contrast, the methanol extract of P. sajor caju exhibited the most potent overall antimicrobial effect. Conclusion: The choice of solvent significantly influences the antimicrobial activity of oyster mushrooms. Oyster mushrooms are natural antimicrobial agents with applications in nutrition, medicine, and agriculture.