The aim of this study was to examine the cytotoxic effect of 10 newly synthesized diazenecarboxamides (diazenes). Using a modified colorimetric MTT assay, their cytotoxicity was determined on 10 human cell lines: cervical carcinoma parental and cisplatin-resistant cells, laryngeal carcinoma parental and cisplatin- and vincristine-resistant cells, glioblastoma parental and cisplatin-resistant cells, breast adenocarcinoma parental and doxorubicin-resistant cells, and mammary carcinoma cells. Results show that diazene JK-279 was most effective, reducing significantly the cell survival of all 10 cell lines examined, including five drug-resistant cell lines. A cytotoxic effect was observed also on nine from 10 cell lines for diazene JK-835. A small reduction in cell survival was obtained (mainly for highest drug concentrations) for diazenes LV-57 and MG-19 on two cell lines, and JK-429 and JK-913 on one cell line. Other diazenes did not demonstrate any cytotoxic activity. The results encourage further research on diazene JK-279 as a potential anticancer drug.