Despite the importance of medicinal plants for the healthcare of local people, the knowledge about medicinal species used in the Pampa biome has been neglected over the years. In this study, an ethnobotanical survey was employed aiming to characterize the species richness and diversity of commercialized medicinal plant species in five cities within the Brazilian Pampa. Additionally, among the listed plants, ten species were selected for in vitro testing of their potential antioxidant activity. A total of 56 plant species belonging to 33 botanical families were listed by the 115 interviewees. No significant difference in commercialized medicinal plant species, and very similar species richness was observed among the cities, indicating that the local knowledge is consistently preserved across the studied cities. According to the biochemical analysis, Sphagneticola trilobata, Malva parviflora and Struthanthus flexicaulis emerged as very promising species for antioxidant activity. Further studies are recommended to advance our knowledge about the richness of medicinal plant species in the Brazilian Pampa, and to assess their therapeutic potential.