4,15‐Diacetoxyscirpenol (DAS) is a mycotoxin primarily produced by Fusarium fungi and occurring predominantly in cereal grains. As requested by the European Commission, the EFSA Panel on Contaminants in the Food Chain (CONTAM) assessed the risk of DAS to human and animal health related to its presence in food and feed. Very limited information was available on toxicity and on toxicokinetics in experimental and farm animals. Due to the limitations in the available data set, human acute and chronic health‐based guidance values (HBGV) were established based on data obtained in clinical trials of DAS as an anticancer agent (anguidine) after intravenous administration to cancer patients. The CONTAM Panel considered these data as informative for the hazard characterisation of DAS after oral exposure. The main adverse effects after acute and repeated exposure were emesis, with a no‐observed‐adverse‐effect level (NOAEL) of 32 μg DAS/kg body weight (bw), and haematotoxicity, with a NOAEL of 65 μg DAS/kg bw, respectively. An acute reference dose (ARfD) of 3.2 μg DAS/kg bw and a tolerable daily intake (TDI) of 0.65 μg DAS/kg bw were established. Based on over 15,000 occurrence data, the highest acute and chronic dietary exposures were estimated to be 0.8 and 0.49 μg DAS/kg bw per day, respectively, and were not of health concern for humans. The limited information for poultry, pigs and dogs indicated a low risk for these animals at the estimated DAS exposure levels under current feeding practices, with the possible exception of fattening chicken. Assuming similar or lower sensitivity than for poultry, the risk was considered overall low for other farm and companion animal species for which no toxicity data were available. In consideration of the similarities of several trichothecenes and the likelihood of co‐exposure via food and feed, it could be appropriate to perform a cumulative risk assessment for this group of substances.