This study aims to assess the phytochemical analysis and evaluate the antioxidant and antimicrobial activities of the methanolic extract obtained from the Algerian Hyoscyamus muticus L. subsp. falezlez leaves of Timimoun region. Methanolic extract of the plant contained the highest quantity of phenolics (148.00 ± 3.07 µg GAE/mg extract) and flavonoids (41.43 ± 0.90 µg QE/mg extract). The High-Performance Liquid Chromatography (HPLC) results showed dominance in the phenolic compounds: orientin, vitexin 2-O-rhamnoside and n-OH-cinnamic acid. Eight metabolites were identified and quantified by Gas Chromatography-Mass Spectrometry (GC-MS) which included five fatty acids, one dicarboxylic acid derivative, one bicyclic hydrocarbon and one fatty acid derivate. The GC-MS analysis revealed that palmitic acid (32.56%), linolenic acid (21.34%) and linoleic acid (11.24%) were the three major components. The methanolic extract showed an antioxidant activity for DPPH, ABTS, reducing power and phenanthroline assays. The strongest antioxidant activity was obtained with phenanthroline assay (value of A0.5 <3.125 µg/mL). The antimicrobial investigation on thirteen microbial strains revealed that the methanolic extract showed low to moderate antibacterial activity against the Gram-positive and negative tested bacteria and no antifungal activity on all the tested fungi. This work suggests the use of leaves from H. muticus L. subsp. falezlez as a source of bioactive compounds with applications in the pharmaceutical, cosmetic and food industries.