In recent years, fungi have been recognized as producers of acetylcholinesterase (AChE) inhibitors, agents important for the prevention of Alzheimer’s disease (AD). This study aimed to examine the AChE inhibitory, the antioxidative and antibacterial activity of two different Schizophyllum commune strains that originated from Serbia (SRB) and Italy (IT). Submerged cultivation of grown mycelia (M) and fermentation broth (F) of ethanol (EtOH) and polysaccharide (PSH) extracts lasted for 7, 14, 21 and 28 days. For AChE activity Ellman method was performed, while for antioxidative activity, sevendifferent assays were conducted: DPPH, ABTS, FRAP, SOA, OH, NO together with total phenolic content. Antimicrobial screen, LC–MS/MS technique and FTIR measurements were performed. Different isolates exhibited different AChE activity, with PSH being the strongest (SRB, M, 28 days IC90 79.73 ± 26.34 µg/mL), while in EtOH extracts, IT stood out (F, 14 days, IC50 0.8 ± 0.6 µg/mL). PSH extracts (7 days) exhibit significant antioxidative activity (AO), opposite to EtOH extracts where 14 and 21days periods stood out. Only tw extracts showed antibacterial activity. Following LC–MS/MS analysis p-hydroxybenzoic and gallic acids were the most abundant phenolics. PSH extracts demonstrated remarkable results, making this study debut and introducing S. commune as a valuable resource of AChE inhibitors.