Acyclovir (ACV)-resistant herpes simplex virus type 1 (HSV-1) causes severe diseases in immunocompromised patients, so identification of new therapies is needed. Interferons (IFNs) are used to treat several other viral infections in the clinic, and IFN-beta and IFN-gamma are known to cooperatively reduce wild-type HSV-1 replication in the corneas of immunocompetent mice. Because IFN-gamma has been shown to exert an antiviral effect mostly through T cells, whether combined IFN treatment can still inhibit ACV-resistant HSV-1 replication, especially in immunocompromised hosts, is unknown. The present study evaluated the efficacy of combined IFN treatment on ACV-resistant HSV-1 mutants. In vitro results showed that IFN-beta acted synergistically with IFN-gamma to inhibit HSV-1 replication in both human and mouse cell lines. Some ACV-resistant mutants were actually hypersensitive to combined IFN treatment. In vivo results showed that topical treatment with a low dose of IFN-beta plus IFN-gamma (200 U each) on mouse corneas efficiently reduced the viral loads by up to 4, 4 and 3 logs, respectively, in the eyes, trigeminal ganglia and brainstems of wild-type and also immunocompromised nude mice infected or co-infected with ACV-resistant HSV-1 in a manner independent of T cells. A highly efficient reduction in HSV acute replication by combined IFN treatment led to a dramatic decrease in subsequent virus reactivation from neural tissues, trigeminal ganglia, brainstems and spinal cords of latently infected mice. Thus, a combination of IFN-beta and IFN-gamma could be a potential treatment for ACV-resistant HSV-1 in immunocompromised patients.