In the search for new derivatives of daunorubicin with high activity and/or the ability to overcome the drug resistance barrier of cancer cells, some new analogs of amidino-daunorubicin, containing the chiral substituent in the formamidine group (-N=CH-N<) at the C-3' position of daunosamine moiety, have been synthesized. In order to estimate the influence of the configuration of the chiral group on the biological properties of the new derivatives of daunorubicin, three chiral amines, namely 1-cyclohexyl-ethylamine, 1-phenylethylamine and N-methyl-l-phenyl-ethylamine, both R and S isomers and their racemates, were used. These new compounds were tested for their cytotoxic activity in vitro against the cells of A549, SW707, T47D and HCV29T cancer lines. The resistance index (RI) values were obtained using the cells of the sensitive LoVo, MES-SA, HL-60 human cancer cell lines, as well as their resistant sublines (LoVo/Dx, MES-SAIDX5 and HL-60/MX2, respectively). All obtained derivatives appeared to be able to overcome the drug resistance barrier of cancer cells.