Mushrooms are valued worldwide for their nutritional, organoleptic and chemical properties. The aim of this study was to determine the chemical composition (free sugars, organic acids, fatty acids, tocopherols and phenolic compounds) and bioactivity of three wild mushrooms (Lactarius piperatus, Lactarius quietus and Lactarius vellereus) from Serbia. Chemical analysis was performed with HPLC-RI and UFLC-PDA (for hydrophilic compounds) and with GC-FID and HPLC-FP (for lipophilic compounds). The analysis of phenolic compounds was performed by UFLC-DAD. Biological activities were evaluated using three different assays (microdilution, TBARS and SRB assays). The results showed that the fruiting bodies were rich in mannitol and trehalose. The main organic acids were oxalic acid and citric acid. As for lipophilic components, stearic, oleic and linoleic acids and β-tocopherol dominated in all the species studied. In addition, the methanolic and ethanolic extracts obtained showed antibacterial, antibiofilm and antioxidant properties. As for cytotoxicity, the extracts were not toxic or only moderately toxic toward different tumor cell lines. According to the results, the selected Serbian mushrooms are a rich source of bioactive compounds, and due to their good biological potential, they can be further exploited as functional ingredients beneficial to human health (antimicrobial agents, antioxidants).