Ants are used as bioindicators because they play diverse ecological roles in terrestrial ecosystems. Their richness, abundance, and broad geographic distribution allow these insects to be used to predict the conditions of environmental conservation. This study aimed to evaluate the bioindicator potential of the ant fauna in Parque Estadual do Turvo, Rio Grande do Sul. Sampling was carried out in two events, in January and November of 2019, using soil and canopy pitfall traps, sardine baits, glucose baits, an entomological umbrella, and a sweeping net. We verified the association of ant species to the sampled strata (soil and leaf litter and vegetation) through an Indicator Species Analysis (ISA) and Principal Component Analysis (PCA). We identified 157 species belonging to 32 genera and eight subfamilies. The subfamily with the most outstanding richness was Myrmicinae, with 70 species, followed by Formicinae, with 40 species. The richest genera in the samples were Camponotus (S = 29), Pheidole (S = 23), and Linepithema (S = 11). Overall, 58 species occurred exclusively in the soil and leaf litter stratum and 23 in the arboreal stratum. Nine species had a significant indication value for the sampled strata, seven for soil and leaf litter, and two for the arboreal strata. The results show the potential of ant fauna as a basis for environmental impact studies and management and conservation plans in the southern Atlantic Forest biome.