The beech mushroom (Hypsizygus marmoreus) is a highly nutritious, edible medicinal mushroom native to East Asia. The present research investigated the impact of different substrates on the metabolite compositions of H. marmoreus mycelia cultivated in vitro. The substrates tested included malt extract agar, malt extract agar enriched with barley malt, and malt extract agar enriched with grape pomace. The study also assessed antimicrobial and antiradical activities of the extracts against gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus), gram-negative bacteria (Escherichia coli, Salmonella typhi, and Pseudomonas aeruginosa), yeasts (Candida albicans, C. tropicalis, and C. parapsilosis), and dermatophytes (Trichophyton mentagrophytes, T. tonsurans, T. rubrum, Arthroderma quadrifidum, A. gypseum, A. curreyi, and A. insingulare). The results revealed that the H. marmoreus mycelia extracts demonstrated antibacterial and antifungal activities against the tested microorganisms. Extracts obtained from the cultivation in substrates enriched with either barley malt or grape pomace exhibited the highest antibacterial activity among all the tested bacterial strains except for P. aeruginosa. The same extracts showed the highest inhibitory effect against C. albicans and C. parapsilosis. Noteworthy, the extract from the mushroom cultivated in the substrate enriched with grape pomace also exhibited remarkable efficacy against T. mentagrophytes and T. tonsurans. Terpenoid and carbapenem compounds could be related to the antimicrobial properties of the extracts from mushrooms cultivated in substrates enriched with grape pomace. In comparison, the higher antiradical properties could be related to the content of indole compounds. In conclusion, growth substrate selection affects the nutritional and medicinal properties of H. marmoreus, making it a valuable contribution to the understanding of the cultivation of this mushroom.