We herein report new 5-substituted uridine derivatives as potent inhibitors of mycobacteria - causative agents of tuberculosis. A series of new 5-alkynyl-substituted uridine derivatives were synthesised via palladium-catalysed Sonogashira cross-coupling reaction of 5-iodo-6-methylpyrimidine base with terminal acetylenes with good yields in DMF at room temperature. It was found that methyl group in C-6 position of pyrimidine ring had no impact on yields of target compounds. All obtained compounds were evaluated for their antimycobacterial activity against Mycobacetrium bovis and Mycobacterium tuberculosis at concentrations of 1-100 µg/ml using MABA test. Synthesized nucleosides showed high antimycobacterial activity against M. bovis and M. Tuberculosis. The MIC50 values of 11 and 13 were similar or close to that of the reference drug rifampicin.