Background Epstein-Barr virus (EBV) is causal in WHO-III nasopharyngeal carcinoma (NPC) and 10% of gastric cancers (GC) worldwide. Since all tumour cells carry EBV, the virus itself is a potential target for therapy. However, EBV expresses few non-immunogenic gene products essential for EBV-DNA maintenance and tumour growth. We developed a cytolytic virus activation (CLVA) therapy, reactivating latent EBV into the lytic cycle, triggering immune recognition and inducing susceptibility to antiviral therapy. Methods CLVA combines a cytostatic drug with valproic acid (VPA) for virus reactivation with (val) ganciclovir (GCV) as an antiviral drug preventing virus replication and killing reactivated tumour cells. CLVA treatment was validated in cell lines and mouse models and in a phase-1/2 clinical trial with Dutch NPC patients refractory to conventional treatment. A similar trial is about to start in Indonesia. Findings In NPC and GC cell lines, various cytostatic drug combinations induce the EBV lytic cycle, which is enhanced by VPA. Addition of GCV resulted in higher viable cell elimination. CLVA therapy eliminated NPC tumours in nude mice more efficiently and completely than single drugs alone. Using 131FIAU, CLVA responsive tumours in mice could be visualised by PET scan and DNA load in blood, mRNA profiling, and in situ staining confirmed in vivo virus reactivation. A phase 1/2 study with six cycles of CLVA treatment showed that CLVA was well-tolerated in 11 patients with end-stage NPC. Patients showed increased viral DNA in blood with lytic mRNA in nasopharyngeal brushings during treatment. Treatment showed a good response in five patients, two patients developed stable disease and in four patients no (early) effect was seen and therapy was ended. Approval for a phase 1/2 trial for NPC was obtained in Yogyakarta, Indonesia. Interpretation CLVA successfully activated EBV from latency in NPC and GC tumour cell lines in vitro and cleared NPC tumours from nude mice. In patients, CLVA was well tolerated and showed a biological effect. This new virus-specific CLVA therapy could open a generic approach for treatment of multiple EBV-associated malignancies.