The aim of this study was to evaluate the use of RNA interference to inhibit herpes simplexvirus type-1 replication in vitro. For herpes simplex virus type-1 gene silencing, three dif-ferent small interfering RNAs (siRNAs) targeting the herpes simplex virus type-1 UL39 gene(sequence si-UL 39-1, si-UL 39-2, and si-UL 39-3) were used, which encode the large sub-unit of ribonucleotide reductase, an essential enzyme for DNA synthesis. Herpes simplexvirus type-1 was isolated from saliva samples and mucocutaneous lesions from infectedpatients. All mucocutaneous lesions’ samples were positive for herpes simplex virus type-1by real-time PCR and by virus isolation; all herpes simplex virus type-1 from saliva sampleswere positive by real-time PCR and 50% were positive by virus isolation. The levels of her-pes simplex virus type-1 DNA remaining after siRNA treatment were assessed by real-timePCR, whose results demonstrated that the effect of siRNAs on gene expression depends onsiRNA concentration. The three siRNA sequences used were able to inhibit viral replication,assessed by real-time PCR and plaque assays and among them, the sequence si-UL 39-1was the most effective. This sequence inhibited 99% of herpes simplex virus type-1 replica-tion. The results demonstrate that silencing herpes simplex virus type-1 UL39 expressionby siRNAs effectively inhibits herpes simplex virus type-1 replication, suggesting that siRNAbased antiviral strategy may be a potential therapeutic alternative.