Our study reports for the first time, over a 12-month period, the seasonal variations of chemical composition, 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. Staphylococcus aureus minimum inhibitory concentration (MIC) values 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 the 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. Correlations between specific compounds and their bioactivity were explored to understand their contributions to the overall efficacy of the RoEO. This comprehensive analysis provides valuable insights into the potential applications and seasonal variability of RoEO from Romania.