We aimed at comparing the effects of domestication and extraction technique on the chemical profiling and antioxidant and antimicrobial activities of Rosmarinus officinalis essential oil (ROEO). This was isolated from wild (WR) and cultivated rosemary (CR) using microwave-assisted extraction (ME) and Clevenger hydrodistillation (CH). Domestication was the main variability source in ROEO constituents, while yield was equally determined by domestication and extraction techniques. Our results revealed important variations, owing to domestication and isolation technique, in terms of ROEO yield (1.10–2.85%), major compounds: α-pinene (14.07–42.03%), camphene (2.26–8.19%), β-pinene (0.35–3.76%), α-terpinene (0.55–2.92%), p-cymene (1.22–4.18%), limonene (0.64–2.79%), 1,8-cineole (31.73–40.72%), β-myrcene (2.09–3.2%), linalool (0.22–1.94%), camphor (12.12–19.66%), borneol (0.53–1.67%), and α-terpineol (1.46–7.45%) as well as minimal inhibitory concentration (MIC, 6.17–15.50 μg/mL), and antioxidant activity (IC50, 2.61–8.58 mg/mL). WR performed better in terms of yield, limonene, cineole, camphor, MIC, and IC50, while the remaining compounds were better expressed in CR. ME displayed high records of ROEO traits except for limonene, camphor, and verbenone (better expressed in CH). Principal component analysis confirmed the obtained findings via the separation of WR, CR, and techniques through the first two components (over 93% of data variability). In conclusion, R. officinalis domestication results in differentiated effects on ROEO traits, fostering a better accumulation of some compounds but reducing yield of other compounds and therefore antioxidant along with antimicrobial activity. ME could be recommended as a green method for ROEO isolation since it was more efficient in terms of the investigated ROEO traits.
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