PLK1 is essential for cell cycle regulation and proliferation, and its elevated expression in prostate cancer is associated with high tumour grade. Therefore, PLK1 inhibition is considered a promising strategy for the treatment of prostate cancer. Here, we identified five compounds (Hits 1-5) targeting the kinase domain (KD) of PLK1 using a combined virtual screening approach. Hits 1-5 all had picomolar (pM) inhibitory potency against PLK1. Notably, Hit-4 showed the strongest inhibitory activity against PLK1 (IC