Application of organic heterocyclic compounds as anti-tumor therapeutics are limited due to therapeutic drawbacks including resistance of tumor cells, non-selectivity of the administered drug towards healthy cells and abundance of unwanted side effects. The aim of our research was to investigate and compare both the antitumor effect and the mechanism of action of newly synthesized, so far untested, chalcone analogue on HeLa, HCT-116 cells and healthy human fibroblast lung cell line (MRC-5). The antitumor efficiency of investigated chalcone analogue was compared to the antitumor effects of dehydrozingerone and cisplatin that were used as referent substances. Cytotoxic and apoptotic effect were evaluated using both MTT test and flow cytometry by Annexin V-FITC/7-AAD staining. The results of our investigation indicated that newly synthesized chalcone analogue H5 expressed more powerful antitumor effect compared to the effects of both dehydrozingerone and cisplatin. Cell death was mediated via apoptotic pathway.