This study investigated the impact of harvest season and extraction method on the yield, composition, and bioactivity of essential oils (EOs) from wild Rosmarinus officinalis L. plants collected in Algeria. Oils were obtained by hydro distillation (HD), steam distillation (SD), and microwave-assisted distillation (MD) across four seasons and characterized by GC–MS. Camphor, α-pinene, camphene, and 1,8-cineole were consistently dominant, with spring oils, particularly those extracted by microwave-assisted distillation, showing the highest enrichment in oxygenated monoterpenes (up to 59.6%). Functional assays revealed clear seasonal variation, whereas spring oils exhibited the strongest antioxidant capacity, with a FRAP value of 4.63 µg/mL, approaching that of the synthetic standard BHA (6.89 µg/mL), alongside notable anti-inflammatory effects. Antimicrobial screening indicated selective inhibition of Escherichia coli and Candida albicans, while Pseudomonas aeruginosa and Bacillus subtilis remained resistant. Acute toxicity evaluation confirmed safety at 2000 mg/kg. These findings demonstrate that ecological timing and extraction strategy critically determine rosemary EO properties and establish quantitative benchmarks for their pharmaceutical and industrial valorization.

This investigation explored how seasonal variation affects the phytochemical composition and biological potential of Rosmarinus officinalis L., a widely used aromatic and medicinal plant. Aerial parts collected during spring, summer, autumn, and winter were extracted with ethanol and analyzed using LC-ESI-MS/MS, while total phenolic (TPC) and flavonoid (TFC) contents were determined spectrophotometrically. The extracts were evaluated for antioxidant, anti-inflammatory, enzyme inhibitory, analgesic, antimicrobial, cytotoxic, and photoprotective properties. Major constituents identified in all seasons included luteolin, kaempferol, rutin, and biochanin A. The autumn extract contained the highest phenolic (353.21 ± 4.05 µg GAE/mg) and flavonoid (190.11 ± 5.65 µg QE/mg) levels. Antioxidant assays revealed that the autumn extract had the strongest DPPH radical scavenging activity (IC₅₀ = 24.72 ± 0.16 µg/mL), while the spring extract exhibited the greatest reducing power (A_0.5 = 7.62 ± 0.30 µg/mL). The winter extract demonstrated superior anti-inflammatory activity (IC₅₀ = 28.60 ± 2.84 µg/mL), exceeding the reference drug diclofenac. Only the spring extract inhibited urease (IC₅₀ = 62.26 ± 0.58 µg/mL) and moderately inhibited α-amylase. All seasonal extracts showed notable photoprotective potential, with SPF values between 25.18 and 32.46, well above the recommended minimum. The spring extract also presented strong analgesic activity and no acute toxicity up to 2000 mg/kg. Antimicrobial effects were weak, limited to slight inhibition of Staphylococcus aureus, while moderate cytotoxicity was observed against MCF-7 and MDA-MB-231 breast cancer cells. Overall, seasonal variation significantly influenced the chemical profile and bioactivities of R. officinalis, with autumn and spring identified as the most suitable harvesting periods for pharmaceutical and cosmetic applications.