Fungus ability to respond to environmental changes allows their adaptation to a wide variety of conditions such as pH, temperature, light, nutrient availability, chemicals, and competition among species. Temperature and pH can affect fungal growth as well as their metabolism. Knowing these factors on fungus species is relevant for the development of biotechnological applications and process optimization to produce biomass and enzymes. This study aimed to evaluate Lentinus crinitus mycelial growth and laccase production at different initial pH and temperatures of the cultivation medium. L. crinitus U15-9 reduced laccase production with pH increase with maximum production at pH 6 (39 U/mL) whereas L. crinitus U9-1 increased laccase production with pH increase with maximum activity at pH 7 (25.8 U/mL). Neither of the strains had mycelial growth significantly affected by initial pH in the culture medium. The greatest laccase production occurred at 28 ºC for U15-9 (50 U/mL) and U9-1 (41.5 U/mL). An increase in U9-1 mycelial biomass occurred when the temperature went up from 25 to 28 ºC whereas for U15-9 it was from 28 to 37 ºC; at 37 ºC, U15-9 produced 225% more mycelial biomass than at 28 ºC, and 108% more mycelial biomass than U9-1 at 37 ºC. The variation of responses to environmental stimuli between the strains makes evident that intra-specific variations are common in basidiomycetes. Our findings highlighted the best conditions of pH and temperature to produce L. crinitus laccase and make evident how different strains respond distinctly to cultivation conditions.