A new N-acetylsulfanilylation series of uridine have been synthesized in good yield using direct acylation method and afforded the 5'-O-N-acetylsulfanilyluridine. In order to obtain newer products, the 5'-O-N-acetylsulfanilyluridine derivative was further transformed to a series of 2',3'-di-Oacyl derivatives containing a wide variety of functionalities in a single molecular framework. The chemical structures of the newly synthesized compounds were confirmed on the basis of their FTIR, 1 H-NMR spectroscopy, physicochemical properties and elemental analysis. All the synthesized uridine derivatives were tested for their in vitro antibacterial activity against six human pathogenic bacterial strains and for comparison standard antibiotic Ampicillin was also determined. The study revealed that the selectively acylated derivatives 5'-O-N-acetylsulfanilyl-2',3'-di-O-lauroyluridine and 5'-O-N-acetylsulfanilyl-2',3'-di-O-pivaloyluridine showed highest inhibition against Staphylococcus aureus and Bacillus cereus, respectively. We also observed that the introduction of hexanoyl, decanoyl, lauroyl, myristoyl and pivaloyl groups, the antibacterial functionality of the compound uridine increases. Another noteworthy observation was that the uridine derivatives * Corresponding author. S. M. A. Kawsar et al. 233were found comparatively more effective against Gram-positive microorganisms than those of Gram-negative microorganisms. In addition, the test chemicals were also tested for cytotoxicity by brine shrimp lethality bioassay and compounds showed different rate mortality with different concentrations.