Epstein-Barr virus (EBV) is a ubiquitous human ɣ-herpesvirus implicated in various malignancies, including Burkitt’s lymphoma and gastric carcinomas. In most EBV-associated cancers, the viral genome is maintained as an extrachromosomal episome by the EBV nuclear antigen-1 (EBNA1). EBNA1 is considered to be a highly stable protein that interacts with the ubiquitin-specific protease 7 (USP7). Here, we show that pharmacological inhibitors and small interfering RNA (siRNA) targeting USP7 reduce EBNA1 protein levels. The USP7 inhibitor GNE6776 altered EBNA1 protein interactions, including disrupting its ability to bind to USP7. GNE6776 also inhibited EBNA1 binding to EBV oriP DNA and reduced viral episome copy number. GNE6776 selectively inhibited EBV ⁺ gastric and lymphoid cell proliferation in cell culture and slowed EBV+ tumor growth in mouse xenograft models. Transcriptomic studies revealed that USP7 inhibition differentially affected EBV ⁺ cancer cells compared to EBV ^- cells with a significant effect on chromosome segregation and mitotic cell division pathways. Our findings indicate that USP7 inhibition perturbs EBNA1 stability and function and can be exploited to target EBV ⁺ cancer cells selectively.