Lactarius sanguifluus is part of the diverse mycological heritage in the Koudiat Taifour forest (a site of biological and ecological interest in the northern region of Morocco), although this fungus is in danger of extinction in this forest. The main objective of the research reported here was to perform an advanced chemical analysis of a methanolic extract from L. sanguifluus and to investigate its biological activities. Spectrophotometric determination of the chemical compounds present in the extract showed that this fungus contains significant amounts of phenolics and flavonoids [18.71–18.81 mg GAE per g of dry methanolic extract (dme)], moderate quantities of ascorbic acid and tannins [3.50–3.86 mg/g of dry weight (dw)], and low levels of β-carotene and lycopene (0.11–0.10 mg/g dme). The phenolic compounds were identified and quantified via LC–MS, and the results showed that p-hydroxybenzoic acid (134.10 µg/g dw) and paraben acid (85.35 µg/g dw) were the main phenolic compounds in the sample. GC–MS analysis of the methanol extract led to the identification of more than 77 chemical compounds in the sample, which were categorized into five main groups: sugars (48.15%), fatty acids (20.66%), amino acids (11.92%), organic acids (9.30%), and alcohols (8.42%). The antioxidant activity of the fungal methanol extract was demonstrated using three methods: the DPPH assay (IC50: 2.045 mg/ml), the β-carotene bleaching test (IC50: 0.56 mg/ml), and the ferric reducing antioxidant power assay (FRAP; RC50: 0.85 mg/ml). The antimicrobial activity was evaluated using microdilution methods. Antifungal susceptibility tests indicated that the extract was able to inhibit the growth of five fungal pathogen species. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) ranged from 1 to 16 mg/ml for Epidermophyton floccosum, Trichophyton rubrum, and Microsporum canis, and from 21 to 64 mg/ml for Candida albicans and Aspergillus fumigatus. Likewise, antibacterial tests showed that the extract inhibited the growth of Escherichia coli and Staphylococcus aureus at concentrations ranging from 21 to 64 mg/ml for the MIC and at 64 mg/ml for the MFC. Considering their nutritional and nutraceutical value, fruiting bodies of L. sanguifluus and extracts from them could be used as nutritional supplements and as natural antioxidant and antimicrobial agents in food preservation and human health.