Cancer is a rapidly growing disease of current era. Continuous research is going on in the direction to develop effective molecules for the treatment of the cancer. The new compounds such as 2,5-diaryl/heteroaryl-1,3,4-oxadiazoles were synthesized and evaluated for anticancer activity. The new 1,3,4-oxadiazole derivatives were obtained by the treatment of aromatic carboxylic acids, with different hydrazide derivatives in the presence of phosphorus oxychloride. Elemental analysis, IR and NMR spectral data confirmed the structures of the compounds. To analyze the antitumor effect of these new compounds, in vitro treatments were performed on two standardized human tumor cell lines, HT-29 (colon adenocarcinoma) and MDA-MB-231 (breast adenocarcinoma), treated with different concentrations of the compounds for different periods of time. The cytotoxic activity of the new compounds was assessed by the colorimetric MTS test. Flow cytometry assay was used to evaluate how compounds influence some processes such as the cell cycle or apoptosis in tumor cells. The analysis of the results shows that the compound with pyridine ring presented the best antitumor profile of the series, probably due to the presence of the pyridine ring next to the oxadiazole structure, acting differently on the two tumor cell lines. The obtained results support the antitumor effect of the analyzed compounds and encourage the extension of the study on other tumor cells in order to improve the anticancer activity and reduce the toxicological risks of the obtained compounds.