Satureja macrostemais 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 leavesSatureja macrostemaand 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 againstEscherichia coliandStaphylococcus aureuswas 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, withtrans-piperitone epoxide (46%),cis-piperitone epoxide (22%), and piperitenone oxide (11%) as more abundant compounds. Likewise,S. macrostemaEOs showed an antioxidant activity of DPPH = 82%, with 50% free radical scavenging (IC50) = 7 mg/mL and TEAC = 0.005, an antibacterial effect againstE. coliof 73% inhibition, and 81% overS. aureusat 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 onS. macrostemashows 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.