Purpose <p><i>Ferula</i> spp. is a significant aromatic and medicinal herb with beneficial medical characteristics in folk medicine, such as anticancer, antioxidant, and antimicrobial properties. This research sought to characterize the chemical profile of the Oleo-Gum-Resin extract gathered from <i>Ferula pseudalliacea</i> roots, a rare plant species of Iran, and to evaluate its biological activities.</p> Methods <p>Chemical constituents of the oleo-gum-resin extract were analyzed using GC-MS/FID. Antimicrobial efficiency was assessed against 11 pathogenic microorganisms using the disc diffusion method and determining the minimum inhibitory concentration (MIC). A DPPH radical scavenging assay was also accomplished to evaluate the antioxidant activity of the extract.</p> Results <p>The Oleo-Gum-Resin contains 34 different bioactive terpenoids and terpene hydrocarbons, with a trace amount of sulfide compounds. The main compounds identified were Bulnesol (31.23%), τ-cadinol (17.83%), and liguloxide (13.82%). The most intense inhibitory effect of the Oleo-Gum-Resin was observed against seven bacterial and two fungal strains to different degrees. <i>S. epidermidis</i> and <i>P. aeruginosa</i> were resistant to the oil. The extract showed a significant DPPH radical scavenging with an IC₅₀ of 41.2 ± 0.2&#xa0;µg/mL compared to the BHT.</p> Conclusion <p>Our findings highlight that the oleo-gum-resin extract of <i>F. pseudalliacea</i> roots is a valuable source of sesquiterpenoides. The extract exhibited a promising antibacterial property against both Gram-positive and Gram-negative bacteria. Additionally, it demonstrated notable DPPH antioxidant activity. Therefore, the oleo-gum-resin of <i>F. pseudalliacea</i> roots could offer a great possibility for developing new types of natural pharmaceutical ingredients.</p>