Introduction: Prostate cancer is the main cause of cancer related deaths in men worldwide. Previous studies have suggested that omega-3 fatty acids reduce cell viability in tumour cells, whereas omega-6 fatty acids increase clonogenicity. Nevertheless, other reports have shown controversial results. Objective: Evaluate cytotoxicity, genotoxicity and clonogenicity in a prostate cancer derived human cell line (PC-3), treated with fatty acids omega-3: α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA); omega-6: linoleic acid (LA) and arachidonic acid (AA); omega-9: oleic acid (OA). Methods: The tests included (a) cytotoxicity assays by MTT and Trypan Blue; (b) genotoxicity evaluation by the sister-chromatid exchanges technique (SCE) and the DNA-comet assay; and (c) in vitro clonogenic assay of six fatty acids in prostate cancer cell (PC-3) at different concentrations (25 µM, 50 µM, 100 µM and 150 µM). Results: The cell viability by MTT data showed ≤ IC50 values for the omega-3 EPA and DHA and omega-6 AA fatty acids at the two major concentrations (100 µM and 150 µM). Moreover, the same fatty acids viability values dropped to 0 % with Trypan Blue test. EPA and DHA showed genotoxic effect and a clonogenic cell decrease (p<0,01). The latter test also revealed a viability diminishment for LA and AA, suggesting different mechanisms of action of fatty acids on cell membrane. Conclusion: The in vitro evaluation revealed that EPA, DHA and AA reduce the clonogenicity and cell viability of prostate tumour cells and cause genotoxicity in prostate tumour derived PC-3 cells.