The prognostic significance of tumour-infiltrating lymphocytes (TILs) in patients with diffuse large B-cell lymphoma (DLBCL) remains controversial. Furthermore, the possible impact of regulatory T cells (Tregs) on survival in DLBCL is still unknown. We performed a retrospective study on the immunohistochemical expression of cytotoxic cells and Tregs, and their correlation with survival in 195 DLBCL patients. Patients with a small number of cytotoxic T-cell intracytoplasmic antigen-1 (TIA-1)+ T cells (≤260 cells/mm tumour area; n = 52) had significantly better outcome than patients with a large number (>260 cells/mm; n = 143); progression-free survival (PFS) at 5 years was 67% vs. 50% (P = 0·03) and overall survival (OS) was 73% vs. 57% (P = 0·03). In multivariate analysis, the low TIA-1+ group still had a better PFS (relative risk 0·75, 95% confidence interval 0·31–0·99; P = 0·05). The number of forkhead box protein 3 (FOXP3)+ Tregs had no influence on PFS (P = 0·89) or OS (P = 0·75). These results suggest that immunohistochemical analysis of cytotoxic T cells at time of diagnosis could provide additional prognostic information. The lack of correlation between the number of FOXP3+ cells and survival could possibly indicate that tumour-infiltrating Tregs are of less clinical importance in DLBCL. However, these findings need to be explored in functional studies.