Prostate cancer is the second most common cause of cancer among men in Australia and worldwide. More than 1.1 million cases of prostate cancer worldwide were recorded in 2012, accounting for around 8% of all new cancer cases and 15% of cancer cases in men. Prostate cancer also accounted for 15% of the total burden of cancer in Australian men in 2012. Even with the advanced treatments developed to manage this cancer disease, the prognosis for survival in prostate cancer patients at a metastatic stage is poor. Therefore, there is a high demand for the discovery of new target-specific anti-prostate cancer drugs. This study focuses on the discovery of natural products with potential anti-prostate cancer activity. A cytotoxicity assay on the human PC3 cells, which are androgen insensitive prostate cancer cells with a highly metastatic potential, was developed to screen a subset of the Nature Bank lead-like enhanced fraction (LLEF) library. High throughput screening of 13,720 natural product LLEFs against PC3 cells and further retest against the human neonatal foreskin fibroblast (NFF) non-cancer cells resulted in five hit fractions (belonging to four different biota) showing selective inhibition against PC3 cells and four hit fractions (belonging to four different biota) showing potent cytotoxicity against both PC3 and NFF cells. Subsequent isolations of six active biota (two sponges Pipestela candelabra and Ancorina geodides, one tunicate Cnemidocarpa stolonifera, one alga Ptilonia australasica, and two plants Austrobaileya scandens and Cephalotaxus oliveri) led to the identification of 16 new natural products together with 29 known compounds.