Abstract: A series of ten pyrimidine nucleosides modified in 2′ position with azide or amine group was tested for the antibacterial, antifungal and cytotoxic activity. The cytotoxic effect was determined on three cancer (CCRF-CEM, MCF7, HeLa) and one normal (HEK293) cell lines, while antibacterial activity was evaluated on five bacterial strains. Among others, 2′-azido-2′deoxycytidine and 2′-amino-2′-deoxycytidine exhibited the strongest antiproliferative activity at 200 μM concentration, decreasing the viability of CCRF-CEM cells to 33 ± 1 and 36 ± 2%, respectively. Newly synthesized 2′-amino-2′-deoxythymidine exhibited cytotoxic effect exclusively toward HeLa cancer cell line, but not toward the normal HEK293 cells. Also, investigated compounds did not exhibit any antibacterial or antifungal activity at a concentration of 40 mM. The obtained results suggest that the presence of cytosine base is desirable for the appearance of cytotoxic effect, while the structural variations of the sugar ring play a minor role. Future modification of 2′-amino-2′-deoxythymidine could be a promising way to obtain more active anticancer substances.