In Eastern Europe, wild mushrooms are widely collected in mountain areas and used for their medicinal properties or as healthy foods. This study aimed at determining the chemical composition (nutritional value, free sugars, organic acids, phenolic compounds, fatty acids and tocopherols) and bioactive properties (antioxidant, antimicrobial and antiquorum sensing) of wild Polyporus squamosus (Huds.) Fr from Romania. The results indicate that the fruiting bodies of P. squamosus are rich in carbohydrates (74.22 g per 100 g dw) and proteins (18.7 g per 100 g dw). Trehalose was the main free sugar, while malic acid was the organic acid detected in the highest amount (2.21 g per 100 g dw), and p-hydroxybenzoic acid was the main phenolic compound. Among tocopherols, β-tocopherol was the most abundant form (114.7 μg per 100 g dw). Additionally, regarding the fatty acids' pattern, polyunsaturated acids represent more than 57% of all fatty acids, followed by monounsaturated fatty acids (24.96%). The highest measured antioxidant effect of P. squamosus extract was found using the TBARS inhibition assay (EC = 0.22 mg mL), followed by the β-carotene/linoleate assay (EC = 1.41 mg mL). A minimal inhibitory concentration of the tested extracts was obtained between 0.61-20.4 mg mL, while the bactericidal effect was achieved between 1.2-40.8 mg mL. Antibiofilm potential was obtained at all tested concentrations, and subinhibitory concentrations of the extract exhibited an antiquorum effect and reduced the formation of P. aeruginosa pili, which all together influenced the virulence of this bacterium. Due to the investigated bioactivities and compounds of P. squamosus and its well-balanced nutritional profile, this mushroom can be further used as a medicinal ingredient based on its antioxidative and antimicrobial potential.