The exact biological functions of individual DNA polymerases still await clarification and therefore appropriate reagents to probe their respective functions are required. In the present study, we report the development of a highly potent series of human DNA polymerase λ and β (pol λ and β) inhibitors based on the rhodanine scaffold. Both enzymes are involved in DNA repair and are thus considered as future drug targets. We expanded the chemical diversity of the small-molecule inhibitors arising from a high content screening and designed and synthesized thirty novel analogs. By biochemical evaluation, we discovered twenty-three highly active compounds against pol λ. Importantly, ten of these small-molecules selectively inhibited pol λ and not the homologous pol β. We also discovered fourteen small-molecules that target pol β and found out that they are more potent than known inhibitors. We also investigated whether the discovered compounds sensitize cancer cells towards DNA-damaging reagents. Thus, we co-treated human colorectal cancer cells (Caco-2) with the small-molecules inhibitors and hydrogen peroxide or the approved drug temozolomide. Interestingly, the tested compounds sensitized Caco-2 cells to both genotoxic agents in a DNA repair pathway-dependent manner.