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La Rioja province annually produces approximately 75,000 tons of agricultural residues and derived materials from agro-industrial activities, which could potentially be incorporated into oyster mushroom cultivation. This study aimed to evaluate the viability of this lignocellulosic biomass as a substrate for the cultivation of edible mushrooms belonging to the genus Pleurotus. Initially, the mycelial growth of two species (P. ostreatus and P. djamor) was assessed by formulating combinations of local substrates. Experimental crops were grown employing the most promising substrates, which were subsequently selected for chemical characterization. It was found that both strains exhibited maximum mycelial growth in the substrate formulated with jojoba leaf litter. A comparison of the two strains revealed no direct correlation between mycelium growth and productive performance. The highest biological efficiency (BE) values were obtained when P. ostreatus was cultivated in treatments combining jojoba leaf litter and grape pomace with olive pomace. Furthermore, these treatments showed suitable chemical properties and were formulated from problematic waste generated in large quantities in the region without proper processing and disposal methods. In this context, there is potential to ensure a continuous supply of this lignocellulosic biomass for cultivating these mushroom species over an extended period of time, thus providing a sustainable alternative for these regional by-products.