Two commercial oyster mushroom species, Pleurotus florida PF05 (PF) and Pleurotus sajor-caju PS08 (PS), were cultivated on wheat straw (WS), corn cob (CC) and combination of both (CC+WS) as basal substrates. These substrates were deliberately supplemented with 5, 10, 20 and 40% of dairy spent wash (DSW) as additive. The influence of DSW on cultivation phases of both mushrooms' lifecycle and other parameters such as mycelial growth rate, ergosterol production, mycelial run, pin head formation, yield, biological efficiency (BE) and protein content in fruitbodies was evaluated. Different DSW concentrations significantly (P< 0.05) exhibited continuous variations over different parameters. In contrast to the response of spent wash on PS, PF was superior in every phase and parameter. The lower concentrations up to a certain level unexpectedly influenced the cultivation phases of both mushrooms. The fastest growth rate (6.6 mm day −1 ) and highest ergosterol (437.00 μg g −1 ) were registered at 20% DSW with the combination of CC+WS as basal substrate. The earliest mycelial run and pin head formation by PF were recorded within 17 and 21 days at 10% supplementation of WS substrate. The maximum 2,173.62 g fruit body yield and 108.68% BE were obtained from PF at 10% DSW supplementation to CC+WS substrates. In addition, the highest content of protein (32.9 mg g −1 ) was achieved in the set of CC+WS with 20% DSW added. The results for each parameter of cultivation in supplemented sets were more enhanced compared to unsupplemented sets. The progression of yield response and biological efficiency was noted as flush-I>flush-II>flush-III with considerable differences. These results suggest the use of DSW at low levels for increasing fruit body yield and BE of Pleurotus mushrooms.