Objective: Derivatives of 1,2,4-triazole are very important in the pharmaceutical industry. Some drugs, including nucleoside analog ribavirin, are available in clinical therapy. However, ribavirin has a number of significant drawbacks, prompting the search for compounds with a more favorable therapeutic index among its structural counterparts. In this study, we synthesized several derivatives of 5-phenyl-1,2,4-triazole-3-thione and enzymatically glycosylated them into 2-deoxyribosides. Molecular docking was used to investigate how a triazole with two hydrophobic substituents could bind to the active site of E. coli purine nucleoside phosphorylase. Methods: The process involves the synthesis of α-D-ribose-1-phosphate from uridine by E. coli uridine phosphorylase (UP). The resulting α-D-ribose-1-phosphate and heterocyclic compound underwent enzymatic glycosylation by E. coli purine nucleoside phosphorylase (PNP) to produce the desired product. Results and Discussion: Previously synthesized 5-phenyl-1,2,4-triazole-3-thione (I) was reacted with alkyl iodide or alkyl bromide in the presence of potassium carbonate in dry acetone. In this way, methyl, ethyl, and propyl derivatives were synthesized. All the compounds were substrates for E. coli purine nucleoside phosphorylase, so enzymatic synthesis of their deoxyribosides was performed. Subsequently, compounds (I–IV) were docked using the SwissDock web service. The Attracting Cavities docking algorithm and the E. coli PNP protein model with 7-deazahypoxanthine and sulfate as ligands (PDB 5IU6) were used. The antiherpetic activity of the synthesized bases and nucleosides was investigated. Conclusions: New 5-phenyl-1,2,4-triazole-3-thione 2-deoxyribosides with bulky substituents at position 3 were synthesized using an enzymatic transglycosylation reaction. A molecular docking study of the 1,2,4-triazole derivatives with two hydrophobic substituents suggested possible modes of their binding to the active site of E. coli purine nucleoside phosphorylase. Both cytotoxicity towards Vero E6 cells and antiviral activity increase with increasing length of the substituent at position 3 in 1,2,4-triazole.