Nucleoside derivatives are important therapeutic drugs and are the focal point in the ongoing search for novel, more potent drug targets. In this study, a new series of pyrimidine nucleoside i.e., uridine (1) derivatives were synthesized via direct method and evaluated for their antimicrobial potential activity. The title compound uridine (1) was treated with triphenylmethyl chloride in pyridine to give the 5´-O-(triphenylmethyl)uridine derivative (2), which was subsequently derivatized to create a series of 2´,3´-di-O-acyl analogs containing a wide variety of functionalities in a single molecular framework. In vitro antimicrobial functionality tests were determined against both human and plant pathogens by disc diffusion and food poisoned techniques. The chemical structures of the synthesized compounds were confirmed on the basis of their spectral, analytical, physicochemical data. The antimicrobial results indicated that the synthesized derivatives exhibited moderate to good antibacterial and antifungal activity; in particular, they were found to be more effective against fungal phytopathogens than against human bacterial strains. Compounds 7, 9, and 14 were of particular interest as they exhibited noteworthy antifungal and antibacterial properties. In vitro MTT assays revealed that compound 9 was effective against Ehrlich’s ascites carcinoma (EAC) cells, resulting in 7.12% and 1.34% cell growth inhibition at concentrations of 200 and 6.25 µg/ml, respectively. The IC50 value for compound 9 was rather high and found to be 1956.25 µg/ml. Structure-activity relationship (SAR) studies were also conducted to predict structural and pharmacokinetic properties. The findings of this study indicate that the different uridine derivatives are potentially useful antimicrobial agents for the advancement of future pharmaceutical research.