The research aims to characterize ethnomedicinal mushroom Lentinus squarrosulus isolates through morphological and molecular approaches. The nutritional requirements and cultural preferences required for the growth of L. squarrosulus were studied in order to maximize the production and its potential values. Morphological features of Lentinus sp. isolates were observed, and molecular methods were performed using DNA sequencing techniques to identify at species level. Culturing the isolates was carried out on potato dextrose agar for assessing cultural traits, colony diameter, mycelial dry weight, in addition to assess the impact of various growth factors like carbon and nitrogen sources, pH, and temperature on mycelial growth. Five isolates of L. squarrosulus were studied in that LS3 demonstrated best growth parameters, with potato dextrose agar as most conducive medium amended with 4% sorbitol and 4% mannitol as optimal carbon sources. Furthermore, maintaining pH levels between 6.0 to 7.0 and temperatures ranging from 25°C to 30°C optimized mycelial growth. The results highlight the crucial significance of comprehending the growth requirements of this mushroom species to maximize its cultivation potential effectively. Further exploration of these findings may unveil behavioral aspects of L. squarrosulus, including alterations in enzyme profiles and phenolic content, potentially fostering applications in biotechnology.