•Large-scale international survey of a protected riparian forest in Central Europe.•Every biotic threat needs their own measures.•Tree diversity negatively affects fungi leave damage. In a changing world, riparian forests are coming under more and more stress from biotic threats. This impacts all stages from saplings to older trees. One of the most dominant biotic threats are alien species and eruptive pest species. Sustainable silvicultural methods are required to reduce the risk of such biotic threats. We investigated the influence of climate, site, tree, and silvicultural characteristics on leaf damage by insect pests or fungal pathogens, herbaceous alien plant species, browsing, and the abundance of native tree saplings. Transects across the Danube-Mura-Drava biosphere reserve from Austria in the northwest to Serbia in the southeast, each containing 7–8 plots, were established to assess tree and site characteristics, regeneration, as well as fungal and insect leaf damage. We found that the horizontal stand structure affected insect leaf damage in different ways. Insect leaf damage was positively correlated with fungal leaf damage and tree height, and negatively correlated with intensity of forest management (from natural forests to coppice/plantations), temperature and precipitation during the vegetation season. Fungal leaf damage was negatively associated with intensity of forest management, and negatively with public ownership and tree species richness. Browsing was positively influenced by the number of saplings. The abundance of alien herbaceous plant species was negatively associated with distance to the watercourse, mean tree height, mean temperature of the vegetative period (MTVP), competition level, and the number of trees per layer, and positively associated with the DBH.