The tumor microenvironment of classical Hodgkin lymphoma (cHL) is clearly responsible for the maintenance of the malignant Hodgkin-Reed-Sternberg (HRS) cells, and Epstein-Barr virus (EBV) has been shown to play a role in this immune evasion. EBV can increase the migration of CD4(+)CD25(+)FOXP3(+) lymphocytes, named regulatory T cells (Tregs). In this study, we assessed the distribution and biological significance of Tregs in patients with cHL. Tissue microarrays were constructed using diagnostic biopsies available in 130 cHL patients and stained with CD4, CD8, CD25, and FOXP3 antibodies. For the present study, only cHL patients whose histology could be confirmed and EBV association established were studied. From the 130 cHL patients selected for this study, 56 were classified as EBV-related and 74 EBV non-related cHL. There were no association between clinical characteristics and the expression of Tregs. However, higher levels of Tregs correlated with EBV presence on HRS cells (p = 0.02), although it did not influence event-free survival (EFS) and overall survival (p = 0.98 and p = 0.59, respectively). This study demonstrates that Tregs expression correlates with EBV presence in HRS cells and has no impact on survival of patients with cHL. Further studies investigating the mechanisms in which EBV recruits Tregs to the tumor microenvironment will contribute not only to our understanding on the pathogenesis of cHL but also to the development of new therapeutic strategies.