DNA polymerase beta (Pol beta) is an error-prone enzyme whose up-regulation has been shown to be a genetic instability enhancer as well as a contributor to cisplatin resistance in tumor cells. In this work, we describe the isolation of new Pol beta inhibitors after high throughput screening of 8448 semipurified natural extracts. In vitro, the selected molecules affect specifically Pol beta-mediated DNA synthesis compared with replicative extracts from cell nuclei. One of them, masticadienonic acid (MA), is particularly attractive because it perturbs neither the activity of the purified replicative Pol delta nor that of nuclear HeLa cell extracts. With an IC50 value of 8 microM, MA is the most potent of the Pol beta inhibitors found so far. Docking simulation revealed that this molecule could substitute for single-strand DNA in the binding site of Pol beta by binding Lys35, Lys68, and Lys60, which are the main residues involved in the interaction Pol beta/single-strand DNA. Selected inhibitors also affect the Pol beta-mediated translesion synthesis (TLS) across cisplatin adducts; MA was still the most efficient. Therefore, masticadienonic acid sensitized the cisplatin-resistant 2008C13*5.25 human tumor cells. Our data suggest that molecules such as masticadienonic acid could be suitable in conjunction with cisplatin to enhance anticancer treatments.