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Background Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV) infection. The gut microbiome can influence outcomes of viral infections but the potential links among the gut microbiome, EBV infection and NPC remain unclear.Objective To characterise gut microbiome alterations in EBV-associated NPC, evaluate microbiome-based diagnostic performance (alone and in combination with EBV markers), and explore associations between microbial features, EBV DNA burden, prognosis and the tumour microenvironment.Design We conducted a large-scale shotgun metagenomic study including 516 patients with EBV-associated NPC and 263 healthy controls. Microbiome dysbiosis, functional pathways and associations with plasma EBV DNA were assessed. Species-level markers were used to build a random forest classifier for NPC diagnosis, and performance was evaluated alone and in combination with EBV-specific markers. Survival analyses were performed to identify microbial features associated with NPC-related mortality and relationships with an immune-suppressive tumour microenvironment were explored.Results NPC was characterised by gut microbiome dysbiosis, including depletion of short-chain fatty acid–producing species and reduced butanoate metabolism, which were significantly associated with plasma EBV DNA. A random forest classifier based on species-level markers distinguished NPC from controls with an area under the curve (AUC) of 0.917; performance improved to an AUC of 0.984 when combined with EBV-specific markers. Specific microbial species were associated with NPC-related mortality and prognostic microbial features were linked to an immune-suppressive tumour microenvironment.Conclusion EBV-associated NPC is associated with distinct gut microbiome and functional alterations that correlate with plasma EBV DNA. Microbial markers show strong diagnostic potential, particularly when integrated with EBV-specific markers, and prognostic microbial features may be linked to an immune-suppressive tumour microenvironment, supporting a potential role of the gut microbiome in NPC tumourigenesis.