Burkitt lymphoma (BL) is a germinal center-derived B-cell neoplasm. Due to the aggressiveness of this neoplasm, treatment must be started quickly. Despite innovation in the pharmaceutical industry, BL has high recurrence and mortality rates. Thus, this study aimed to evaluate the cytotoxic effect of an imidazopyridine (DSH65) on BL lineage cells, and to investigate its main mechanisms of cell death. Initially, the concentration-and-time-response curves were evaluated in Daudi and PBMC cells. DSH65 showed IC values of 50.14 ± 3.14 µM, 17.62 ± 0.87 µM, and 13.12 ± 0.71 µM at 24, 48, and 72 h in Daudi cells, respectively, with an IC of 74.08 ± 1.62 µM in PBMCs at 24 h. DSH65 did not cause significant hemolysis and induced apoptosis, confirmed by morphological changes and phosphatidylserine externalization by flow cytometry. The results obtained for the compound DSH65 show that the mechanism of action is related to an intrinsic apoptosis, with an increase in Bax/Bcl-2 ratio, loss of Δψm and caspase-3 activation. Incubation with the DSH65 also reduced survivin and Ki-67 expression, increased ROS production, and caused G2/M phase cell cycle arrest. These findings suggest DSH65 as a promising candidate for further drug development for BL treatment.