This study evaluated the influence of seasonal variation on the yield and composition of essential oil of Lippia origanoides occurring in the Middle Rio Amazonas, Brazil, and the impact on its antimicrobial potential. The average oil yield was 1.7% ± 0.2% in the rainy season and 1.6% ± 0.3% in the dry season. Some correlations with climatic parameters were observed. The major components were carvacrol (rainy, 43.5% ± 1.9%; dry, 41.4% ± 2.04%), thymol (rainy, 10.7% ± 1.1%; dry, 10.6% ± 0.9%), p-cymene (rainy, 9.8% ± 0.7%; dry, 10.0% ± 1.4%) and p-methoxythymol (rainy, 9.6% ± 0.8%; dry, 10.4% ± 1.4%). It was found that the antibacterial activity of L. origanoides against Staphylococcus aureus and Escherichia coli was little influenced by the changes in oil composition due to seasonal variation. Against S. aureus, the oil Minimum Inhibitory Concentration (MIC) value was 1.25 μL/mL over ten months. Against E. coli, the oil MIC values ranged from 0.15 μL/mL to 0.31 μL/mL in different months of the year. The Minimum Bactericidal Concentration (MBC) value was 2.5 μL/mL against S. aureus and 1.25 μL/mL against E. coli. The results suggest that the antimicrobial activity identified in the oil remain unchanged for the full year, allowing its medicinal use without any risk of loss or absence of the active principles of the plant.