Background: Understanding patterns of species diversity can only be achieved by long-term research and the integration of taxonomical, ecological, and behavioral data. Here, we studied the diversity and ecology of ants of Iguazu National Park (INP), Argentina, using six sampling techniques. DNA barcodes were used to uncover cryptic diversity, test species/morphospecies delimitation accuracy, and link unidentified male and queen specimens with their worker caste. Results: INP houses 195 described ant species and an additional 49 morphospecies. Leaf litter sampling and pitfall traps were the most efficient sampling methods, while surface baiting revealed the prevalence of large predatory species at different times of the day. Comparing baiting to other sampling methods provided information on species co-existence and the presence of possible dominance hierarchies among ant species. We obtained the DNA barcodes of 312 specimens from 124 species (51% of the ants of INP). Our analyses evidenced a clear barcode gap in all species but two, with an average distance to the nearest neighbour of 15.75%, almost eight times larger than the mean distance to the furthest conspecific (2.07%). Eighty-three percent of the sequence clusters obtained with different clustering algorithms (ABGD, RESL, TCS) matched the reference species or morphospecies, while 10% highlighted possible cryptic diversity. In terms of efficacy, this barcode library allowed a correct identification in more than 94% of the species/morphospecies, and to assign a species name to 69% of the unidentified males and queens. Significance: This study evidences that DNA barcodes are a valuable tool for identifying the ants of the Atlantic Forest, a global diversity hotspot. Furthermore, our project provides a framework for understanding the ecology and the taxonomic diversity of the ants of this region, including the identification of currently undescribed reproductive castes and the discovery of possible cryptic species.