Tree cultivation is widely stimulated due to the provision of environmental services by sequestering atmospheric carbon, but forestry is a potential danger to biodiversity, mainly when cultivation replaces originally non-forested landscapes. Due to the rapid expansion of afforestation in grassland ecosystems, we used environmental descriptors and structure of amphibian and reptile communities as a tool to assess the possible effects of forestry on wildlife. We used pitfall traps with drift fences in Eucalyptus afforestation and native grasslands of the Pampa biome from southern Brazil. Amphibians and reptile species richness was lower in afforestation than in native grasslands. Furthermore, the reptile community found in afforestation was a poor subset of the community recorded in native grasslands. Indeed, the structure of communities for both amphibians and reptiles in afforestation was highly dissimilar from the native grasslands, with major environmental descriptors that influenced this difference being related to the structure of undergrowth vegetation, incident light, and sodium concentration in soil. Amphibians and reptiles proved to be an efficient tool to assess the potential impacts effects of afforestation/forestation since the replacement of natural grassland ecosystems by eucalypt afforestation caused detectable negative effects on the diversity of herpetofauna. Biodiversity conservation strategies in non-forest ecosystems are urgently needed and should encompass policies to restrict the expansion of forestry on the remaining native grasslands.