A. mellea fruiting bodies collected from nature were chemically characterized and shown to be rich in carbohydrates (81.25 g per 100 g dw), ash, fat and proteins (8.84 g per 100 g dw, 1.97 g per 100 g dw and 1.81 g per 100 g dw, respectively). Mannitol was the main free sugar while malic acid was the most abundant organic acid. δ-Tocopherol was the dominant form of tocopherols with 42.41 μg per 100 g dw. Polyunsaturated fatty acids were predominant, followed by saturated and monounsaturated fatty acids. A methanolic extract prepared from these samples was tested for antioxidant, quorum sensing and antimicrobial assays, as well as for its cytotoxicity effects. The extract showed antimicrobial activity against all tested microorganisms, including Candida albicans. Furthermore, when tested at sub-MIC concentration, it showed reduction of virulence factors and biofilm formation against Pseudomonas aeruginosa. The extract also exhibited antioxidant activity and did not show toxicity against tumor and non-tumor cells. Due to the observed bioactive properties and compounds of the honey mushroom and its well-balanced nutrients, this mushroom emerges as an interesting functional food and a source of nutraceuticals with applications in different diseases based on antioxidant and antimicrobial effects.