Lentinus squarrosulus (Mont.) Singer, a wild edible macrofungus, possesses confirmed organoleptic, nutritional, and medicinal attributes. During the exploration of fungal biota of Punjab, Pakistan, L. squarrosulus was collected under the Morus alba from University of the Punjab, Lahore. For the domestication of this wild significant species, its culturability and cultivation potential was assessed by using different synthetic culture media and substrates. Among these different media used, maximum cultural growth was observed on the malt-extract agar (MEA) medium at 35°C followed by the potato-dextrose agar (PDA), compost extract agar (CEA), glucose-peptone agar (GPA), and Sabouraud-dextrose agar (SDA). Cultured strains on MEA medium were used to produce spawning material on wheat, sorghum, and barley grains. Sorghum grains at 35°C were found to be the best combination for spawn production of this fungus. Yield and biological efficiency were investigated on a variety of substrates. A mixed substrate of wheat straw and tea waste at 30°C showed the optimum yield. Tea-waste medium was used as the casing material and proved very effective. Mineral analysis of the wild and cultivated strain was carried out and revealed that both forms were enriched with macronutrients. These results showed that the L. squarrosulus strain from Pakistan possesses good nutrient and growth potential and its domestication can compete with nutritional and medicinal properties of commonly growing mushrooms.