Background <p>Epstein–Barr virus (EBV) positivity is a potential predictive biomarker for immune checkpoint inhibitors (ICIs) in gastric cancer patients, but its value in first-line ICI-based chemotherapy remains unclear. This study aimed to evaluate the predictive value of EBV positivity in patients treated with first-line nivolumab plus chemotherapy. </p> Methods <p>This single-center study included advanced gastric cancer patients treated with first-line nivolumab plus chemotherapy (n = 293). Patients with EBV positivity treated with chemotherapy alone (n = 12) served as the control group. EBV positivity was confirmed by <i>in situ</i> hybridization.</p> Results <p>Among patients treated with nivolumab plus chemotherapy, 18 (6.1%) had EBV-positive tumors, and these were associated with high PD-L1 combined positive score (CPS) expression levels. Progression-free survival (PFS) and overall survival (OS) tended to be more favorable in those with EBV-positive tumors. Multivariate analysis of patients treated with nivolumab-chemotherapy revealed that EBV positivity, combined with PD-L1 CPS ≥ 5, was an independent factor for PFS. In patients with EBV-positive tumors, nivolumab-chemotherapy was associated with significantly favorable PFS and OS compared to chemotherapy alone. Similar results were observed in the subgroup with PD-L1 CPS ≥ 5. However, survival outcomes did not differ between patients treated with nivolumab plus chemotherapy versus chemotherapy alone in the subgroup with PD-L1 CPS &lt; 5.</p> Conclusion <p>EBV positivity predicts favorable survival outcomes in patients with gastric cancer treated with nivolumab plus chemotherapy. The benefit of nivolumab plus chemotherapy over chemotherapy alone for patients with EBV positivity appears to be associated with high PD-L1 expression levels.</p>