The chemical profiles of Rosmarinus officinalis L. essential oils, collected from five distinct geographical regions in Palestine, were determined using GC-MS. The major phytochemical classes of R. officinalis EOs were monoterpene hydrocarbon (24.81–78.75%) and oxygenated monoterpenoids (19.01–73.78%), with 1,8-cineole (4.81–37.83%), α-pinene (13.07–51.36%), and camphor (11.95–24.30%) being the most abundant components of the studied oils. Using the DPPH assay, the antioxidant activity of EOs revealed that EO from the Jenin region had the highest antioxidant activity, with an IC50 value of 10.23 ± 0.11 µg/mL, followed by samples from Tulkarm (IC50 = 37.15 ± 2.3 µg/mL) and Nablus (IC50= 38.9 ± 0.45 µg/mL). With MICs of 12.5, 12.5, 6.25, 6.25, and 6.25 µg/mL against MRSA, S. aureus, E. coli, K. pneumonia, and P. vulgaris, respectively, the EO extracted from the Jenin region of Palestine had the greatest antibacterial activity. Furthermore, EOs from Jenin and Nablus demonstrated stronger anti-candida action than the pharmaceutical formulation Fluconazole, with MICs of 0.781, 0.781, and 1.56 µg/mL, respectively.