The European Commission requested EFSA to evaluate whether a series of dyes are covered by the ‘Guidance on methodological principles and scientific methods to be taken into account when establishing Reference Points for Action (RPAs) for non‐allowed pharmacologically active substances present in food of animal origin’ and to which group they should be attributed according to this guidance. Although these substances are not registered for use in food‐producing animals in the European Union, they may be used illegally in aquaculture for their antimicrobial properties. It was concluded that acriflavine, 3‐aminoacridine, aminoacridine, basic blue 7, brilliant green, leucobrilliant green, C.I. basic blue 26, chloranil, crystal violet, leucocrystal violet, dichlone, ethyl violet, methylene blue, new methylene blue, Nile blue, pararosaniline base, proflavine, proflavine hydrochloride, rhodamine 6G and trypan red are covered by the guidance document and belong to group I. A toxicological screening value of 0.0025 μg/kg body weight per day is applicable. Azure blue and potassium permanganate were excluded from the evaluation due to their inorganic nature.