Tropane alkaloids (TA) are secondary metabolites occurring in several plant families, including Solanaceae. The most studied TA are (–)-hyoscyamine and (–)-scopolamine, which is in contrast to the (+)-enantiomers formed naturally. The racemic mixture of (–)-hyoscyamine and (+)-hyoscyamine is called atropine. In 2013, the EFSA CONTAM Panel carried out an acute dietary exposure assessment for the sum of (–)-hyoscyamine and (–)-scopolamine (group Acute Reference Dose (ARfD) 16 ng/kg body weight (bw)), and identified a potential concern for toddlers. This scientific report provides a more extensive acute dietary human exposure assessment of TA using 39,725 analytical results on 31 TA sampled between 2009 and 2016 in 13 European countries. Most of the analytical results (95%) were left-censored (i.e. below the limit of detection or below the limit of quantification). High concentrations were reported for atropine and scopolamine in tea and herbal infusions, cereal bars and spices. The mean and 95th percentiles (P95) acute dietary exposures to the sum of atropine and scopolamine were highest in infants, toddlers and other children. Under the upper bound (UB) assumption, the group ARfD exceeded for the sum of atropine and scopolamine at the mean level in toddlers and other children, and at the P95 in all age classes. Under the lower-bound (LB) assumption, the group ARfD was exceeded for atropine at the P95 in toddlers, and for the sum of atropine and scopolamine at the P95 in toddlers and other children. Very large differences were observed between the LB and UB estimated exposure levels across all age classes. Overall, among processed foods, the main contributors to the exposure to the sum of atropine and scopolamine were, at the UB, bread and cereal-based foods, and, at the LB, tea and herbal infusions for all age classes.