Illegal wildlife poisoning is a global threat for biodiversity, yet the magnitude of its impact on ecosystems is largely underestimated as most of poisoning episodes remain undetected. Here, we conducted a large-scale field experiment to better understand the real dimension of the illegal wildlife poisoning in terms of composition and number of species and abundance of impacted individuals, as well as the ecological factors driving it. We used camera traps to monitor simulated poison baits placed in 25 study areas in SW Europe and applied Good–Turing theory to estimate the richness of species of the entire assemblage (observed plus undetected). We recorded 3095 individuals from 39 vertebrate species that consumed 94 % of the baits (N = 590). Yet, using sample completeness to estimate the entire species assemblage yielded a total of 47 species exposed to illegal poisoning. The observed assemblage included different trophic and functional groups (from lizards and snakes to apex species among birds and mammals), as well as a 38 % of threatened and near threatened species (according to Spanish and Portuguese vertebrate red list and UICN list). The size (weight) of the bait outstands as a reliable predictor of the number of species (0–8 species/bait, mean = 2) and individuals (0–99 individuals/bait, mean = 5) susceptible to poisoning. The habitat where the bait was placed modulated the abundance of individuals affected (greater in open than in closed habitats). Type of bait and habitat drove the compositional variation of species. Our approach enables uncover entire species assemblages prone to illegal poisoning and their ecological drivers associated, advancing the understanding of the impact of wildlife poisoning in ecosystems.