Hippomarathrum scabrum L. is an endemic medicinal plant in Turkey; however, there have been few studies investigating the phytochemistry and biological properties of these plants has not been investigated. The aim of this work is to determine the chemical composition of different extracts (extracts obtained by using supercritical carbon dioxide extraction, accelerated solvent extraction, homogenizer-assisted extraction, microwave-assisted extraction, and ultrasound-assisted extraction from Hippomarathrum scabrum L., and evaluate their biological properties. The analysis revealed that 5-O-caffeoylquinic acid, rutin, and isorhamnetin 3-O-rutinoside were the main bioactive compounds. The extract obtained by accelerated extraction contains the highest concentration of 5-O-Caffeoylquinic acid (7616.74 ± 63.09 mg/kg dry extract) followed by the extract obtained by homogenizer-assisted extraction (6682.53 ± 13.04 mg/kg dry extract). In antioxidant tests, all extracts expressed significant antioxidant activity. Also, cytotoxic and anticancer effects of these plant extracts were detected in the human prostate cancer cell line. Intrinsic apoptotic genes were up-regulated and anti-apoptotic genes were down-regulated in human prostate cancer cells after inhibition concentration dose treatment. The findings are promising, and suggest the use of these plant extracts could be used as natural sources with different biological activities, as well as anticancer agents.