Our study reports for the first time, over a 12-month period, the seasonal variations in chemical composition and antibacterial and antioxidant activity of Rosmarinus officinalis L. essential oil (RoEO) from Southwestern Romania (Oltenia region). To analyze the constituents of RoEO, a comprehensive gas chromatography/mass spectrometry (GC/MS) method was employed. The analysis aimed to identify and quantify the various components by comparing their mass spectra with reference spectra from the National Institute of Standards and Technology (NIST) Library 2020. The minimum inhibitory concentration (MIC) values of Staphylococcus aureus minimum were determined using the microdilution method (96-well plates). The antioxidant activity was analyzed using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and hydrogen peroxide (H2O2) radical scavenging assays. This analysis provided a detailed profile of RoEO’s constituents, revealing significant monthly variations. Key compounds, such as camphor, eucalyptol, α-pinene, camphene, and α-myrcene, were quantified, alongside lesser-studied constituents like β-pinene, α-terpinene, linalool, terpinolene, and carvacrol. Comparisons were made with a reference sample from Tunisia. Oxygenated monoterpenes reach the highest concentration (56.82–66.94%), followed by monoterpene hydrocarbons (30.06–40.28%), sesquiterpene hydrocarbons (0.90–2.44%), and oxygenated sesquiterpenes (0.02–0.23%). Camphor was found in high concentrations ranging from 29.41% to 40.03%. 1,8-Cineole was another dominant compound, ranging from 13.07% to 16.16%, significantly lower compared to the Tunisian reference (52.77%). α-Pinene ranged from 11.36% to 19.33%, while α-myrcene ranged from 1.65% to 3.08%. Correlations between specific compounds and their bioactivity were explored to understand their contributions to the overall efficacy of RoEO. This comprehensive analysis provides valuable insights into the potential applications and seasonal variability of RoEO from Romania.
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