Nucleoside analogs are frequently used in the control of viral infections and neoplastic diseases. However, relatively few studies have shown that nucleoside analogs have antibacterial and antifungal activities. In this study, a fused pyrimidine molecule, uridine, was modified with various aliphatic chains and aromatic groups to produce new derivatives as antimicrobial agents. All newly synthesized uridine derivatives were analyzed by spectral (NMR, FTIR, mass spectrometry), elemental, and physicochemical analyses. Prediction of activity spectra for substances (PASS) and in vitro biological evaluation against bacteria and fungi indicated promising antimicrobial capability of these uridine derivatives. The tested compounds were more effective against fungal phytopathogens than bacterial strains, as determined by their in vitro antimicrobial activity. Cytotoxicity testing indicated that the compounds were less toxic. In addition, antiproliferative activity against Ehrlich ascites carcinoma (EAC) cells was investigated, and compound6(2′,3′-di-O-cinnamoyl-5′-O-palmitoyluridine) demonstrated promising anticancer activity. Their molecular docking againstEscherichia coli(1RXF) andSalmonella typhi(3000) revealed notable binding affinities and nonbonding interactions in support of this finding. Stable conformation and binding patterns/energy were found in a stimulating 400 ns molecular dynamics (MD) simulation. Structure–activity relationship (SAR) investigation indicated that acyl chains, CH3(CH2)10CO-, (C6H5)3C-, and C2H5C6H4CO-, combined with deoxyribose, were most effective against the tested bacterial and fungal pathogens. Pharmacokinetic predictions were examined to determine their ADMET characteristics, and the results in silico were intriguing. Finally, the synthesized uridine derivatives demonstrated increased medicinal activity and high potential for future antimicrobial/anticancer agent(s).