Abstract Polo-like kinase 1 (PLK1) inhibitors have had limited antitumor efficacy as single agents, and a focus of current efforts is on combination therapies. We initially confirmed that the PLK1 specific inhibitor onvansertib (ONV) could enhance responses to a PARP inhibitor (olaparib) in prostate cancer xenografts. To identify more effective combinations we screened a library of bioactive compounds for efficacy in combination with ONV in LNCaP prostate cancer cells, which identified a series of compounds including multiple AKT inhibitors. We confirmed in vitro synergy between ONV and the AKT inhibitor ipatasertib (IPA) and found that the combination increased apoptosis. Mechanistic studies showed that ONV increased expression of the anti-apoptotic protein SURVIVIN, and that this was mitigated by IPA. Studies in three PTEN deficient prostate cancer xenograft models showed that co-treatment with IPA and ONV led to significant tumor growth inhibition compared to monotherapies. Together these in vitro and in vivo studies demonstrate that the efficacy of PLK1 antagonists can be enhanced by PARP or AKT inhibition, and support further development of these combination therapies.