The valorization of lignocellulosic residues and unconventional plant biomass offers a promising strategy for sustainable mushroom production within a circular bioeconomy framework. This study evaluated the suitability of four substrates, namely wheat straw, Typha latifolia (cattail) biomass, Ampelodesmos mauritanicus biomass, and coffee waste, for the cultivation of Pleurotus ostreatus. The effects of two concentrations of glucose and potassium oxide on fungal colonization and fruiting performance were investigated under laboratory-scale conditions. The hypothesis was that substrate composition and nutrient supplementation would differentially influence mycelial development and mushroom production. Mycelial growth was evaluated through daily measurements of colony expansion, whereas fruiting performance was assessed through mushroom yield. Substrate type significantly affected both mycelial growth and yield. A. mauritanicus showed high potential for mycelial colonization, particularly after supplementation with glucose (20 g/L) or potassium (10 g/L). For fruiting body production, potassium supplementation improved cattail performance, with the highest yield obtained with 20 g/L potassium. Glucose and potassium supplementation also enhanced the productivity of A. mauritanicus. All tested fertilization concentrations increased P. ostreatus fructification from 1.5 to 8%, except when glucose was added to the coffee waste substrate. The tested supplementation rates had substrate-dependent effects, confirming that nutrient addition did not produce a universal response. Mineral analysis of harvested mushrooms revealed differences in nutritional composition among treatments. These results highlight the potential of cattail and A. mauritanicus biomass as alternative substrates for P. ostreatus cultivation. Further economic feasibility studies are required before scale-up applications.