Background: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease with poor outcome. Based on the cell of origin (COO), it is classified into germinal center B (GCB) and non-GCB subtypes. The interleukin 17A (IL-17A) and tumor-associated macrophages in the tumor microenvironment have been involved in modulation of tumor immunity in colon cancer and non-Hodgkin lymphoma. Enhancer of zeste homolog 2 (EZH2) has been associated with poor prognosis of several tumors. The aim of the current work is evaluation of the effect of IL-17A, CD68, and EZH2 protein expressions and COO classification in predicting response to R-CHOP as well as patient outcome in DLBCL. Patients and methods IL-17A, CD68, and EZH2 proteins were evaluated by immunohistochemistry in 60 cases of DLBCL. Moreover, tumors were classified into GCB and non-GCB types based on Hans algorithm using CD10, BCL6, and MUM1 immunohistochemistry. The pattern of protein expression was correlated to patients’ characteristics, response to R-CHOP, and patient survival. Results Overexpression of IL-17A protein was significantly associated with involvement of more than one extranodal site, high lactate dehydrogenase level, performance score (PS) more than or equal to 2, as well as high International Prognostic Index (P<0.001, P=0.002, P<0.001, P=0.004, and P<0.001, respectively). Both EZH2 protein overexpression and non-GCB type were significantly associated with advanced tumor stage, higher lactate dehydrogenase level, PS, as well as International Prognostic Index (P<0.001). However, CD68 immunopositivity was not related to any of the studied parameters (P≥0.05). We found that cases with IL-17A or EZH2 overexpression or non-GCB phenotype were highly resistant to R-CHOP (P<0.001). Moreover, they were associated with short-term disease-free survival, progression-free survival, and overall survival (P<0.05). CD86 immunoreactivity was significantly associated with longer disease-free survival (P=0.016), but not progression-free survival, overall survival, or response to therapy (P<0.05). Conclusion IL-17A and EZH2 proteins and COO classification could be used for risk stratification of newly diagnosed patients with DLBCL and predicting their prognosis and response to therapy. Moreover, a panel of markers should be used for examining tumor-associated macrophages instead of CD68 alone.