Abstract A comparative study of phytochemicals content and biological properties of eight Rosmarinus officinalis (rosemary) populations (RO1–RO8) collected in different areas of Tunisia was carried out. Two of the main rosemary constituents, rosmarinic and carnosic acids, were quantified by an NMR technique. Carnosic acid content was higher than that of rosmarinic acid. The non-polar constituents were examined by GC and GC–MS. Total phenols and flavonoids content were also determined in order to discuss the possible correlation between these phytochemicals and bioactivity. Antioxidant activity was investigated through different in vitro assays. Sample RO3 from a sub-humid area showed the highest potency in 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay (IC50 of 6.74μg/mL) while RO5 from arid area exhibited the highest radical 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) scavenging activity (IC50 of 5.92μg/mL). The most active sample in Ferric Reducing Antioxidant Power (FRAP) assay was RO4 (62.21μM Fe(II)/g), followed by RO1–RO3 and RO8 harvested in sub-humid areas with values in the range 49.61–58.72μM Fe(II)/g. Extracts were able to inhibit acethylcholinesterase (AChE) and butyrylcholinesterase (BChE). Sample RO5 exhibited a promising AChE inhibitory activity (IC50 of 4.47μg/mL). Thus, we suggest rosemary extracts as a functional food ingredient for preventing AD.