Objective: Epithelial ovarian cancer (EOC) has a poor prognosis because of high recurrence rate due to chemoresistance, and there is an unmet need to find new therapeutic drugs. Fenbendazole (FZ) is an anti-helminthic drug acting as microtubule-disrupting agent, and previous studies suggested this drug to have anticancer effects. However, the poor water solubility of FZ limits its potential for cancer therapy. In this study, we combined FZ with poly (lactic-co-glycolic acid) (PLGA) nanoparticles to obtain hydrophilicity. We further investigated the anticancer effect of FZ with different drug delivery method on EOC cell lines in both in vitro and in vivo. Methods: EOC and normal epithelial cell lines were treated with FZ, and cell survival was assessed using MTT assay. The effect of FZ on tumor growth in orthotopic mouse xenografts of EOC cell line was examined. To improve systemic delivery of FZ by converting fat-soluble drugs to hydrophilic, we prepared FZ-encapsulated PLGA nanoparticles (FZ-PLGA-NPs). We investigated the preclinical efficacy of FZ-PLGA-NPs with in vitro and in vivo models including patient-derived xenograft (PDX) for human EOC. Results: In cell proliferation experiments, FZ showed non-specific cytotoxicity not only in both chemosensitive (A2780, HeyA8, SKOV3ip-1) and chemoresistant (A2780-CP20, HeyA8-MDR, SKOV3-TR) EOC cells but also in normal epithelial cell lines (IOSE, 283T, HUVEC). However, in xenograft mouse models of EOC using HeyA8 and HeyA8-MDR, there were no effect on tumor reduction when treated with either 1mg or 10mg of FZ orally. When administered intraperitoneally, FZ was not absorbed but aggregated in intraperitoneal space. We hypothesized that FZ was not absorbed into the body due to its poor water solubility, so we synthesized FZ-PLGA-NPs to obtain water solubility and enhance drug absorption. Loading efficiency of FZ in FZ-PLGA-NP was 89%. In cell proliferation experiments of FZ-PLGA-NP in EOC cell lines showed similar results with FZ alone showing cytotoxic effect among all cells. In in vivo experiments of xenograft mouse model with HeyA8 and HeyA8-MDR, FZ-PLGA-NP (containing 1mg of FZ) intravenous injection demonstrated significantly reduced tumor weight compared to control group. FZ-PLGA-NP also showed anti-cancer effect in PDX model of chemosensitive high grade serous ovarian cancer. Conclusion: This study demonstrated anticancer effect of FZ to EOC cells in water soluble form of FZ-PLGA-NPs. This result suggests that nanoparticle mediated drug delivery system could be a new strategy for drug repositioning.