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The abundant biomass growth of aquatic macrophytes in wetlands is one of the major concerns affecting their residing biota. Moreover, the biomass degenerates within the wetlands, thereby causing a remixing of nutrients and emission of greenhouse gases. Therefore, it is crucial to find sustainable methods to utilize the biomass of aquatic macrophytes devoid of environmental concerns. The present study investigates the utilization of the biomass of three aquatic macrophytes, including the lake sedge (CL: <i>Carex lacustris</i> Willd.), water hyacinth (EC: <i>Eichhornia crassipes</i> Mart. Solms), and sacred lotus (NL: <i>Nelumbo nucifera</i> Gaertn.) to produce oyster (<i>Pleurotus ostreatus</i> var. <i>florida</i>) mushrooms. For this purpose, different combinations of wheat straw (WS: as control) and macrophyte’s biomass (WH) such as control (100% WH), CL50 (50% WH + 50% CL), CL100 (100% CL), EC50 (50% WH + 50% EC), EC100 (100% EC), NL50 (50% WH + 50% NL), and NL100 (100% NL) were used for <i>P. florida</i> cultivation under controlled laboratory conditions. The results showed that all selected combinations of wheat straw and macrophyte biomass supported the spawning and growth of <i>P. florida</i>. In particular, the maximum significant (<i>p</i> < 0.05) growth, yield, bioefficiency, proximate, and biochemical parameters were reported using the WH substrate followed by CL, NL, and EC biomass, which corresponds to the reduction efficiency of the substrate parameters. Therefore, the findings of this study reveal that the biomass of selected aquatic macrophytes can be effectively utilized for sustainable mushroom cultivation while minimizing the risk associated with their self-degeneration.