Natural products represent a rich source of biologically active compounds and an example of molecular diversity with recognized potential in drug discovery. Nowadays, medicinal mushrooms used in folk medicine are being increasingly studied for their successful application in pharmaceutical industries. In this context, Pleurotus species are one such potential mushroom with worldwide acceptance as a delicious food and famed for remarkable medicinal properties. However, till date no reports regarding the bioactivity of the most compatible herbal formulations of this mushroom (infusion and decoction) have been documented. Therefore, the present study has been undertaken to investigate the phytochemical composition and antioxidant properties of three cultivated species of this group namely, P. eous, P. florida and P. ostreatus. Phytochemical composition revealed that the infusion fraction of P. eous is the most enriched fraction having high amount of phenols (13.03 μggallic acid equivalent/mg of extract), flavonoids (3.57 μg quercetin equivalent/mg of extract) and ascorbic acid (16.66 μg/mg of extract). Moreover, free radical scavenging potentials of the corresponding fractions of these mushrooms were tested by different in-vitro systems. Results demonstrated infusion had noticeable scavenging activity in concentration dependent manner while decoction exhibited moderate activity for all three species. Decoction did not show any activity in hydroxyl radical scavenging assay. Together, our findings provide a detailed data explaining beneficial therapeutic properties of aqueous fraction of these Pleurotus species. Additionally, it also can be concluded that in comparison with other studied mushroom, P. eous infusion is superior in terms of antioxidant efficacy. © 2020 DSR Publishers/The University of Jordan. All Rights Reserved.