In an effort to increase the potency and selectivity of earlier identified substrate-based inhibitors of mitochondrial thymidine kinase 2 (TK-2), we now describe the synthesis of new thymidine analogues containing a 4- or 5-substituted 1,2,3-triazol-1-yl substituent at the 3'-position of the 2'-deoxyribofuranosyl ring. These analogues were prepared by Cu- and Ru-catalyzed cycloadditions of 3'-azido-3'-deoxythymidine and the appropriate alkynes, which produced the 1,4- and 1,5-triazoles, respectively. Selected analogues showed nanomolar inhibitory activity for TK-2, while virtually not affecting the TK-1 counterpart. Enzyme kinetics indicated a competitive and uncompetitive inhibition profile against thymidine and the cosubstrate ATP, respectively. This behavior is rationalized by suggesting that the inhibitors occupy the substrate-binding site in a TK-2-ATP complex that maintains the enzyme's active site in a closed conformation through the stabilization of a small lid domain.