Ferula species have been recognized as crucial medicinal plants that are known for producing a variety of sesquiterpenes, such as sesquiterpene lactones and sesquiterpene coumarins. However, guaianolide sesquiterpenes, which are sesquiterpene lactones that contain an alpha‐methylene‐gamma‐lactone moiety, are rare in Ferula spp. Ferula hindukushensis, an endemic species found in Afghanistan, Pakistan, and Iran, belongs to the Ferula genus. In this study, the normal column chromatography technique was employed to fractionate the dichloromethane extract of F. hindukushensis. The structures of two guaianolide sesquiterpenes were identified using various nuclear magnetic resonance (NMR) spectra, such as 13C NMR, 1H NMR, and heteronuclear multiple bond correlation (HMBC), and were compared with the literature interpretation to confirm their structural characteristics. Moreover, the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay was employed to examine the cytotoxicity of guaianolide compounds on various cell lines, including MCF‐7, 3T3, HT‐29, MCF‐10, PC12, and A549. The antioxidant effects of these compounds were evaluated using ferric reducing antioxidant power (FRAP) and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) assays. The findings indicated that the antioxidant activity of the compounds was lower than that of vitamin C at the same concentration. The cytotoxic effects of the compounds varied depending on the cell types. Nevertheless, except for HT‐29, a concentration lower than 125 μg/mL was found to be non‐toxic for all cell lines.