The current research work aims to identify the brown rot fungi Daedalea quercina, grown in Tunisia, using typical molecular tools, and to assess its chemical composition by UPLC-MSe analysis, antioxidant capacity using three different methods (DPPH and ABTS radical scavenging assay and FRAP) and antibacterial activity against a collection of human pathogens. The D. quercina fruiting body hydroalcoholic extract (EtOH-H2O-E) and its ethyl acetic fraction (EtOAc-F) revealed the presence of 25 secondary metabolites including triterpenes such as daedaleanic acid A, carboxyacetylquercinic acid and the 24,31-didehydro-3-(carboxyacetyloxy) quercinic acid, many of which are here reported in D. quercina for the first time. The EtOH-H2O-E of D. quercina displayed the most active DPPH and ABTS scavenging activities (598.38 and 389.20 µg/mL respectively). The antibacterial results indicated that Bacillus subtilis, a Gram-positive species, was the most sensitive to the studied extracts with low MIC values (0.03 and 0.06 mg/mL). Both extracts were proven to exhibit antitumor activity towards HeLa cells, and a protective capacity of HUVEC cells in a dose-dependent manner. The EtOH-H2O-E showed the best antitumor activity at concentrations ranging from 25 to 200 μg/ml with protection to HUVEC cells at the same concentrations. To the best of our knowledge, this represent the first chemical and pharmacological characterization of D. quercina from Tunisia, and overall one of the most exhaustive characterization of secondary metabolites in this species. Based on the encouraging obtained data, D. quercina can represent a valuable natural product with pharmaceutical and nutraceutical applications in virtue of its biological activities and its content in potential bioactive compounds.