The objective of this study was to analyze the mycelial growth under different culture conditions and the antimicrobial activity of Lentinus citrinus Walleyn & Rammeloo DPUA 1535 and Neolentinus lepideus (Fr.) Redhead & Ginns DPUA 1536 against some fungi and bacteria. The growth of Lentinus spp. was evaluated in natural and complex media, with and without light, at 25°C and 28°C for 8, 15, and 30 days. To test antimicrobial activity, we used Candida albicans DPUA 1336, Cryptococcus laurentii DPUA 1501, Aspergillus flavus DPUA 1836, Escherichia coli DAUPE 224, and Mycobacterium smegmatis ATCC 607. L. citrinus showed strong growth in potato dextrose and rice meal agars in the absence and presence of light, at 28°C, and over 8, 15, and 30 days. N. lepideus grew well in Sabouraud agar in the absence and presence of light at 25°C for 8 and 15 days. L. citrinus evidenced mycelial growth and high density in potato dextrose agar, although lesser mycelial density was observed in rice bran extract agar. N. lepideus showed less abundant mycelial growth and density in the 3 culture media. Organic mycelial extracts of Lentinus species demonstrated a potential antibacterial and antifungal activity, with selectivity to the test-microorganisms under in vitro growth conditions.