AimsThe characterization of the antitumour activity and chemical identification of the compounds obtained after the isolation of extracellular metabolites of bacteria Raoultella ornithinolytica. Methods and ResultsThe fraction with anticancer activity against the HeLa cell line, T47D and TOV-112D was obtained from the supernatants of R.ornithinolytica culture using ion-exchange chromatography, and separated by Sephadex G-50 medium gel filtration into two subfractions. The obtained compounds were analysed using Fourier Transform-Infrared Spectroscopy, Raman spectroscopy and matrix-assisted laser desorption/ionization MS/MS spectrometry. The antitumour activity of the two subfractions was analysed using 5-bromo-2-deoxy-uridine kit. The subfraction with the highest activity against HeLa cells was identified as Tris-peptide complex. The amino acid sequence of the peptide from the complex was found to be TDAPSFSDIPN and molecular weight was estimated at 14306576Da. Cytotoxic, cytopathic and apoptotic effects in HeLa cells treated with the active complex were observed; however, the cytotoxic effect against normal human skin fibroblasts was minimal. ConclusionsThe Tris-peptide complex from R.ornithinolytica showed selective antitumour activity against the HeLa cell line. Significance and Impact of the StudyThe Tris-peptide complex due to the high selectivity can be used in biomedicine, and its derivatives may contribute to the development of new anticancer compounds.