A series of new 3'-deoxyribosides of substituted benzimidazoles was obtained by the chemo-enzymatic method using genetically engineered purine nucleoside phosphorylase (PNP). In the case of asymmetrically substituted benzimidazole derivatives, a mixture of N1- and N3-regioisomers was formed (confirmed by NMR). The antiviral activity of the obtained compounds against herpes simplex virus 1 of reference strain L2 and a strain deeply resistant to acyclovir in Vero E6 cell culture was studied. 4,6-Difluoro-1-(β-D-3'-deoxyribofuranosyl)benzimidazole (IC = 250.92 µM, SI = 12.00) and 4,5,6-trifluoro-1-(β-D-3'-deoxyribofuranosyl)benzimidazole (IC = 249.96 µM, SI = 16.00) showed significant selective activity against both viral models in comparison to ribavirin (IC = 511.88 µM, SI 8.00).