In vivo antinociceptive and in vitro radical scavenging and cytotoxic activities of Acanthus hirsutus Boiss. aqueous extract were investigated to give a new insight into plant usage in traditional medicine. The extract showed significant antinociceptive activity in acetic acid-induced writhing test in mice after oral application and did not change the hind-leg retraction period in the hot-plate test for any dose applied. In addition, the extract showed radical-scavenging activity against 2,2-diphenyl-1-picryl-hydrazyl, nitric oxide, and superoxide radicals similar to those of standard compounds 3-t-butyl-4-hydroxyanisole, ascorbic acid (vitamin C), and quercetin. The gallic acid equivalent total phenolic content of the plant was found to be 65.4 mg/g dry extract. Cytotoxic activity of the aqueous extract was tested against 3 different cancer cell lines-Hep-2 (human larynx epidermoid carcinoma), RD (human rhabdomyosarcoma), and L20B (transgenic murine L cells)-and 1 noncancerous cell line (VERO) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Through the phytochemical studies, the following compounds were isolated: 3 lignan glucosides [(-)-syringaresinol-di-O-β-glucopyranoside, (-)-medioresinol-di-O-β-D-glucopyranoside, (-)-pinoresinol-4'-O-β-glucopyranoside], 2 benzoxazinoids [2-hydroxy-1,4-benzoxazin-3(4H)-one, (2R)-2-O-β-glucopyranosyl-1,4-benzoxazin-3(4H)-one], 4 phenylethanoid glycosides (acteoside, leucosceptoside A, martynoside, hattushoside), and 2 phenylpropanoid glucosides (sinapyl aldehyde-4-O-β-glucopyranoside, sinapyl alcohol-4-O-β-glucopyranoside). Cytotoxic and radical-scavenging activities of the isolated compounds were also determined. 2-hydroxy-1,4-benzoxazin-3(4H)-one and acteoside were the most active compounds in both experiments.