Following a request from the European Commission to EFSA, the Scientific Panel on Food Additives and Nutrient Sources added to Food (ANS) was asked to provide a scientific opinion on the safety in use of natamycin (E 235) as a food additive, and on the issue of antimicrobial resistance to natamycin. Natamycin is a fungicide of the polyene macrolide group. According to Directive 95/2/EC, natamycin may be used for the surface treatment of semi-hard and semi-soft cheese and dry, cured sausage at a maximum level of 1 mg/dm2 in the outer 5 mm of the surface. The SCF in 1979 considered that the database was adequate to conclude that natamycin does not give rise to safety concern, but inadequate to establish an ADI. JECFA assigned an ADI of 0.3 mg/kg bw/day (1968, 1976, 2002). The Panel considered that the available data are not sufficiently robust for the purpose of deriving an ADI because of the limitations of the present database on natamycin (design of the animal studies, limited number of animals, lack of a carcinogenicity study) and in view of the inadequate description of the human data. The highest potential exposure to natamycin was below 0.1 mg/kg bw/day for children at the 97.5th percentile. Given that natamycin is very poorly absorbed, the Panel considers that this conservative estimate would provide an adequate margin of safety from the effect level seen from the long-term animal studies and the human study used by JECFA to establish an ADI. The Panel considered that the proposed use levels of natamycin are not of safety concern if it is only used for the surface treatment of the rind of semi-hard and semi-soft cheese and on the casings of certain sausages. The Panel concluded that there was no concern for the induction of antimicrobial resistance.