Objective To evaluate in vitro effects of Tagetes minuta L. essential oil (TEO) on L3 Anisakis larvae type 1. Methods In order to evaluate the potential use of Tagetes minuta essential oil against L3 Anisakis larvae three different media were tested: 1) a saline solution (SS); 2) an industrial marinating solution (MS); 3) sunflower seeds oil (SO). For each media and concentrations of TEO (0.1%, 0.5%, 1.0% and 5.0% v/v), 20 parasites were introduced into plastic Petri dishes (diameter 90 mm) and maintained at room temperature. As controls, larvae were maintained without TEO under identical experimental conditions in SS, MS and SO. A total of 900 larvae were tested. The normalized mean viability, LT100, LT50 and the percentage of inactivation at 24 h were calculated. Results In vitro tests revealed a complete inactivation of parasites in saline solution after 2 h with 5% and 1% of TEO. In marinating solution, a complete inactivation of parasites was observed after 4 h at all concentrations used. A slower activity for all TEO concentration was reported in SO. Conclusions The results obtained, showing a strong activity against Anisakis larvae, confirm TEO as a larvicidal agent in the treatment of human anisakidosis and in the industrial marinating process.