| Summary: | <i>Satureja macrostema</i> is a plant that is located in various regions of Mexico and is used in a traditional way against illness. Essential oils (EOs) were obtained from leaves <i>Satureja macrostema</i> and the chemical composition was evaluated by gas chromatography–mass spectrometry (GC-MS). The antioxidant effect of the oil was assayed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and by Trolox Equivalent Antioxidant Capacity (TEAC). In vitro antibacterial activity against <i>Escherichia coli</i> and <i>Staphylococcus aureus</i> was determined using a broth microdilution assay and thin layer chromatography–direct bioautography (TLC-DB) to identify active antibacterial compounds. The EOs analysis showed 21 compounds, 99% terpenes, and 96% oxygenated monoterpenes, with <i>trans</i>-piperitone epoxide (46%), <i>cis</i>-piperitone epoxide (22%), and piperitenone oxide (11%) as more abundant compounds. Likewise, <i>S. macrostema</i> EOs showed an antioxidant activity of DPPH = 82%, with 50% free radical scavenging (IC<sub>50</sub>) = 7 mg/mL and TEAC = 0.005, an antibacterial effect against <i>E. coli</i> of 73% inhibition, and 81% over <i>S. aureus</i> at dose of 100 µL of undiluted crude oil. The TLC-DB assay showed that the most active compounds were derived from piperitone. The comparison with other studies on <i>S. macrostema</i> shows variability in the compounds and their abundances, which can be attributed to climatic factors and the maturity of plants with similar antioxidant and antibacterial activities.
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