Abstract Objectives The aim of this study was to investigate the cytotoxic and antitumour effects of the essential oil from the leaves of Mentha x villosa (EOMV) and its main component (rotundifolone). Methods In-vitro cytotoxic activity of the EOMV and rotundifolone was determined on cultured tumour cells. In-vivo antitumour activity of the EOMV was assessed in sarcoma 180-bearing mice. Key findings The EOMV displayed cytotoxicity against human tumour cell lines, showing IC50 values in the range of 0.57–1.02 μg/ml in the HCT-116 and SF-295 cell lines, respectively. Rotundifolone showed weak cytotoxicity against HCT-116, SF-295 and OVCAR-8 cell lines (IC50 > 25.00 μg/ml). Tumour growth inhibition rates were 29.4–40.5% and 25.0–45.2% for the EOMV treatment by intraperitoneal (50–100 mg/kg/day) and oral (100–200 mg/kg/day) administration, respectively. The EOMV did not significantly affect body mass and macroscopy of the organs. Conclusions The EOMV possesses significant antitumour activity with low systemic toxicity, possibly due to the synergistic action of its minor constituents.