Introduction The traditional use of phyto remedies and natural products may indicate their pharmacological potential. Screening plant extracts to identify their active components has gained momentum with possible beneficial effects for public health. The aim of study was to assess and compare the biological properties of different extracts (methanolic, aqueous, and ethyl acetate) prepared from Acanthus hirsutus (AH), a traditionally used medicinal plant. Methods The phytochemical profile of AH was established via analysis of the total phenolic and flavonoid content, as well as the condensed tannins and gallotannins present in plants collected in the Anatolian region of Turkey. Chemical profiles were confirmed using the high-performance liquid chromatography (HPLC/DAD) method. Antioxidant potential were appraised through the analysis of their free radical scavenging activity, ferrous ion chelating ability, hydroxyl radical scavenging activity, total antioxidant capacity, and inhibition ability of lipid peroxidation. Any cytotoxicity activity of the extracts was assessed through the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay against three different cancer cell lines. Results The HPLC/DAD profile revealed the presence of rutin, p-hydroxybenzoic acid, vanillic acid, chlorogenic acid, and naringenin which were in significant abundance in the extracts. The aqueous extract had the highest antioxidant capacity with 284.33 ± 0.05 μg AAE/g extract, which correlated with the highest total condensed tannins, phenolics, and flavonoid contents (94.14 ± 0.16 mg GAE/g extract, 114.30 ± 0.11 mg GAE/g extract and 61.18 ± 0.43 mg RE/g extract, respectively). Different sensitivities were observed towards the different cell lines used, with the aqueous extract being the less cytotoxic. Conclusion AH proved to be a potent medicinal plant that can be further exploited as a complementary and alternative therapy for the management of oxidative stress related diseases such as cancer, diabetes, and rheumatoid arthritis.