Abstract Nepeta rtanjensis Diklic & Milojevic (fam. Lamiaceae) is an endemic, critically endangered plant, protected by the law in Serbia. Various biological activities have been ascribed to its major constituents, nepetalactones. In this study we describe for the first time cytotoxic activity of N. rtanjensis essential oil (EO), obtained from field cultivated plants, which was found to be especially rich in trans,cis-nepetalactone. MTT assays indicated that after 72 h of treatment the EO exhibited cytotoxic activity on investigated cancer cell lines: HeLa, K562, A549, LS-174 and MDA-MB-231. Normal cell line (MRC-5) was the least sensitive to the treatment and IC50 value for this cell line was not reached within the tested range of EO concentrations (up to 0.1 μL/mL). Analysis of morphological changes of treated cells confirmed the higher sensitivity of tumor cells than normal cells to the tested EO. Application of N. rtanjensis EO resulted in the appearance of morphological changes in tested cancer cell lines characteristic for apoptotic cell death, and induced perturbations of the cell cycle of HeLa cells. In addition, upregulation of Bax and p53, and downregulation of Bcl-2, and Skp2 genes, involved in apoptotic signalling cascades, confirmed an apoptosis-inducing effect of N. rtanjensis EO on HeLa cells. Presented results highlighted the potential of N. rtanjensis EO in anticancer therapy.