Sloe (Prunus spinosa L.) extracts are a good source of natural bioactive compounds, including phytosterols. Phytosterols are known to be applied in the treatment of various prostate diseases. The in vitro antiproliferative activity of sloe ethanolic extracts (flower, leaf, and fruit), collected from three areas in Bosnia and Herzegovina, were investigated against human prostate cancer cell lines PC-3 and DU145 using MTT assay. β-sitosterol, campesterol and stigmasterol were quantified by HPLC-PDA analysis using Symmetry C18 chromatographic column. The results of the analysis proved the presence of phytosterols, mostly β-sitosterol in all extracts. All extracts possess antiproliferative activity. The highest activity against PC-3 and DU145 was gathered from leaf extracts obtained by different extraction methods (microwave-assisted extraction and ultrasound-assisted extraction). To the best of our knowledge, no other studies have presented results on antiproliferative activity of ethanol sloe extracts. Based on these results, further investigation should be recommended on other cancer cell lines as well.