Introduction Euxyl K400 is a widely used preservative in cosmetics, topical drugs, cutting fluids, etc. This antimicrobial product has two active components—methyldibromo glutaronitrile (MDGN), also known as 1,2-dibromo-2,4-dicyanobutane, and 2-phenoxyethanol, in a proportion of 1 to 4.The aim of this study was to assess the prevalence of sensitization to this preservative in cases in our dermatology department over a 5-year period. We also assessed the prevalence of sensitization to the two active components of Euxyl K400 and calculated the agreement with sensitization to Euxyl K400. Material and methods In this retrospective study, we analyzed the medical records of 1,092 patients attended in our skin allergy unit between January 2000 and December 2005.We undertook testing with a standard battery that included, in addition to the allergens recommended by the Spanish Group for Research Into Dermatitis and Skin Allergies (GEIDAC), 0.3% MDGN and 1% 2-phenoxyethanol in Vaseline (Trolab®). Results Only 15 patients developed a positive reaction to Euxyl K400. Of these, 11 were positive to MDGN and 2 to phenoxyethanol. Sensitization to Euxyl K400 was significantly more common in men. The agreement between sensitization to Euxyl K400 and MDGN was good (Kp = 0.68), whereas agreement between Euxyl K400 and phenoxyethanol was poor (Kp = 0.23). Conclusions The prevalence of Euxyl K400 sensitization in this study was 1.4%. Most cases of Euxyl K400 sensitization were associated with the MDGN component, as indicated by the good agreement between the two substances.