Low-intensity ultrasound is a useful method to enhance the delivery of drugs to target cells via a range of mechanisms including the transient formation of micropores in the cell membrane, a process known as sonoporation. The effect of ultrasound on oncolytic herpes simplex virus type-1 (HSV-1) infection in oral squamous cell carcinoma (SCC) was examined. Human SCC cell line SAS and oncolytic HSV-1 RH2, which was deficient in the neurovirulent γ 1 34.5 gene and exhibited cell fusion actions, were used. Cells grown in multi-well plates were infected with HSV-1 and exposed to ultrasound in the presence or absence of microbubbles after an adsorption period. The number of plaques was significantly greater than that of the untreated control. SAS cells were inoculated subcutaneously into nude mice and tumors were produced. Tumors were injected with HSV-1 RH2 with or without microbubbles and then exposed to ultrasound through the covering skin. The amount of the virus in tumor tissues 3 days after the injection was higher in tumors treated with HSV-1 RH2 and ultrasound than in tumors treated with RH2 only. The expression of the HSV-1 antigen was also increased by ultrasound and microbubbles. Tumor growth was suppressed with HSV-1 RH2 in combination with ultrasound, especially with microbubbles. These results indicated that ultrasound increased the efficiency of the HSV-1 infection in SAS cells and nude mouse tumors. This method can potentially be useful to enhance the antitumor effects of oncolytic HSV-1 on head and neck cancer treatment.