Biodiesel obtained from oil extracted from the viscera of tilapia is a viable alternative in the replacement of petroleum fuels. However, during the purification step is performed biodiesel washing water is performed, which generates high effluent pollutant loads due to the reagents used and the very composition of the raw material. This study aims to characterize the fatty acids present in water from washing of the process of purifying biodiesel tilapia (Oreochromis niloticus). Fatty acid compositions were determined using gas chromatography (GC-FID). The results showed that the fatty acids present in greater quantities in the effluent were lauric (C12: 0), followed by myristic (C14: 0), palmitic (C16: 0), oleic (C18: 1), stearic (C18: 0), linolenic (C18: 3), and linoleic (C18: 2) acids. Therefore, the levels of oil and grease found in the rinse water from washing of the oil biodiesel tilapia are far above the allowed values above; thus they do not comply with Brazilian federal regulations.