Pimpinella L., Apiaceae, is one of the largest genera and has a broad spectrum of biological or pharmacological activities including antiproliferative effects against cancer cells. In the present study, the cytotoxic activity of the methanol extracts and sub-extracts of Pimpinella isaurica V.A. Matthews subsp. isaurica was investigated against the cervical epithelial carcinoma cell line (HeLa), the liver carcinoma cell line (HepG2), the breast carcinoma cell line (MCF-7), and the lung carcinoma cell line (A549) by MTT assay. For the chemical profiling, the methanol extract was analyzed for phenolic acids and flavonoids by HPLC-DAD. Mutagenic and biological activities of detected compounds were predicted with in silico methods. P. isaurica subsp. isaurica methanol extract showed potent activity on all tested cell lines according to National Cancer Institute guidelines (IC50 ranges 5.36–15.57 μg/ml). Apigenin-7-O-glucoside (0.098 g/100 g) and luteolin-7-O-glucoside (0.078 g/100 g) were revealed in the most active extract, and these compounds were predicted as nonmutagenic. The cytotoxic activity of P. isaurica subsp. isaurica against cervical, liver, breast, and lung cancer cells and flavonoids in the plant was reported for the first time. Graphical Abstract: [Figure not available: see fulltext.]