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This study describes the chemical composition, antitumor, antioxidant, and antimicrobial activities of the plant <i>Thymus algeriensis</i> Boiss. Essential oils (EOs) were collected in different periods (before, during, and after flowering stage) from the El-Guetfa region, M’sila, Algeria. The EOs extraction was achieved using three distinguishing techniques: hydro (Clevenger trap), steam, and microwave distillations, targeting different aerial parts of the plant (stems, flowers, and leaves). The EOs chemical components were estimated using GC-FID and GC-MS apparatuses. The resulting yield of the extracted oil was moderate and ranged between 0.84 and 1.53% (<i>wt</i>/<i>vol</i>). In total, eighty-five components were identified, in which the oxygenated monoterpenes family formed the main portion, starting from 40.56 up to 70.66%. The obtained essential oil was dominated by five major components that varied from low to quite moderate percentages: camphor (17.45–32.56%), borneol (11.16–22.2%), camphene (7.53–12.86%), 1.8-cineole (5.16–11.21%), and bornyl acetate (3.86–7.92%). The biological results of this oil pointed out that the EOs extracted from the leaves part exposed a weak radical scavenging activity afterward using two well-known antioxidant assays DPPH (IC₅₀ = 8.37 mg/mL) and ABTS (10.84 mg/mL). Meanwhile, this oil presented strong inhibition activity against colon cancer cell line HCT116 (LC50 = 39.8 µg/mL) and a moderate inhibitory against hepatocellular cancer cells HePG2 (LC50 > 100 µg/mL). In addition, this oil antimicrobial activity was quite important against <i>Micrococcus luteus (M. luteus</i>)<i>, Staphylococcus aureus</i> CIP 7625, <i>Escherichia coli</i> ATCC 10536, <i>Saccharomyces cerevisiae</i> ATCC <i>4226</i>, <i>Candida albicans</i> IPA200, <i>Candida tropicalis (Ct),</i> and <i>Candida glabrata (Cg)</i> after using Amoxicillin and Itraconazole as references.