BACKGROUND: The causal relationship between Epstein-Barr virus (EBV) infection and frailty or fatigue remains unclear, despite evidence linking chronic inflammation to these conditions. METHODS: This study utilized a two-sample Mendelian Randomization (MR) framework to investigate the causal relationship between EBV infection and the development of frailty and fatigue. The outcomes assessed were frailty, defined by the Frailty Index (FI), and fatigue, measured through Chronic Fatigue Syndrome (CFS), Malaise and Fatigue (MF), while EBV infection was represented by anti-EBV IgG seropositivity, antibody levels, and a history of infectious mononucleosis. Genetic variants strongly associated with EBV exposure were identified and used as instrumental variables (IVs). Two-sample MR analyses were conducted using the Inverse Variance Weighted (IVW) method, and multivariable MR (MVMR) was applied to adjust for potential confounding, including age. Reverse MR analyses were also performed to explore reverse causality. Sensitivity analyses, including horizontal pleiotropy and leave-one-out tests, were carried out to assess the reliability of the results. RESULTS: A total of 9 GWAS were used to derive summary data for EBV-related exposures and frailty/fatigue outcomes. In multivariable MR, EBV ZEBRA antibody levels were significantly associated with an increased FI score (aβ = 0.026; 95 % CI 0.006, 0.046; P = 0.011) after age adjustment. EBV EA-D showed a significant link with CFS in unadjusted models, but lost significance after age adjustment. EBV VCA p18 and EA-D were associated with MF, with significance remaining for EBV VCA p18 (aOR = 1.25; 95 % CI: 1.01, 1.57; P = 0.046) and EA-D (aOR = 1.38; 95 % CI: 1.00, 1.90; P = 0.049) after age adjustment. The reverse MR analysis revealed negative associations between MF and EBNA-1/ZEBRA antibodies. Sensitivity analyses confirmed robustness with no evidence of pleiotropy or heterogeneity. Secondary analysis further supported the causal associations. CONCLUSION: EBV infection demonstrates causal links to frailty and fatigue, mediated through specific antibody responses. These findings emphasize EBV's role in chronic inflammatory pathways and highlight potential targets for clinical intervention.