Rosmarinus officinalis L. (rosemary) is a common culinary spice and herbal drug, which is used for centuries all over the world. In this present study, apolar to polar fractions of R. officinalis flowers were evaluated for their in vitro antioxidant, antibacterial, cytotoxic, anti-inflammatory and analgesic activities, respectively. Phytochemical compositions of R. officinalis extract fractions were analyzed by GC-MS and LC-MS. The antioxidant capacity of the fractions was evaluated by using the DPPH center dot and ABTS(center dot) methods. The antibacterial potential was determined using the in vitro broth microdilution assay against a panel of human pathogens. The analgesic and anti-inflammatory activities were investigated measuring nitric oxide (NO) and prostaglandin E-2 (PGE(2)) production in LPS-stimulated cells, respectively. In addition, in vitro cytotoxicity of the extract fractions was evaluated on RAW 264.7 murine macrophage cells by using the MTT assay. The constituents of the polar fractions were identified as rosmarinic acid, luteolin, quercetin and apigenin by LC techniques, whereas the n-hexane fraction was analyzed by GC-MS to determine the main volatile components camphor (19.6%), 1,8-cineole (11.7%), verbenone (11.5%), borneol (10.6%), alpha-pinene (5.8%), and linalool (5.7%). According to the bioactivity results, the polar fraction showed the highest antioxidant activity, whereas n-hexane fraction was found to be most effective against Staphylococcus aureus (78 mu g/mL). The n-hexane fraction (100 mu g/mL) reduced the LPS-induced NO and PGE(2) production capability. In conclusion, R. officinalis flower n-hexane and ethyl acetate fractions exhibited remarkable in vitro antibacterial, antioxidant, anti-inflammatory and analgesic activities possibly due to their polyphenol content, to the best of our knowledge for the first time. (C) 2019 SAAB. Published by Elsevier B.V. All rights reserved.