This study was oriented towards the investigation of the biological properties of three wild growing and edible Macrolepiota species (M. mastoidea, M. rhacodes and Macrolepiota procera) from Serbia. The results revealed that the mushrooms have a low caloric value; free sugars such as mannitol and trehalose were identified; oxalic and malic acids were predominant organic acids, while p-hydroxybenzoic and p-coumaric acids were identified as the main phenolic compounds. Also, they were a rich source of polyunsaturated fatty acids, which dominated over monounsaturated and saturated fatty acids. Three isoforms of tocopherols were identified and quantified: α-, β-, and δ-tocopherol. Regarding biological properties, all three species exhibited antioxidant potential, antimicrobial potential and cytotoxic activity within the different tumour cell lines tested. This study indicates that these species are indeed functional foods, due to the fact that they are edible, consumable and hold different pharmacological activities.