Documentation of species occurrences through compilation of checklists is an important component of biodiversity conservation and planning, which are limited in sub-Saharan Africa. As an ecologically diverse country, ant checklist compilation was carried out in Eswatini in order to determine ant diversity and distribution within the country’s major ecological terrestrial biomes. Collections by various sources over the years have resulted in the determination of 148 species from 41 genera in 6 subfamilies, with Myrmicinae being the dominant one with 74 species. Within the terrestrial biomes, the savanna had 77% of the species recorded while grassland and forest biomes had 52.7% and 25% of the species, respectively. Three species, Anoplolepis custodiens (Smith, 1858), Polyrhachis schistacea (Gerstäcker, 1859) and Pheidole megacephala (Fabricius, 1793) were the most widely distributed in the country. There were also the first records of Myrmicaria faurei Arnold, 1947 and Terataner transvaalensis Arnold, 1952 outside of their known ranges in Mpumalanga, South Africa, as well as Camponotus liogaster Santschi, 1932 outside of its known range in KwaZulu-Natal, South Africa. The forty-eight species identified only to genus level illustrates the taxonomic gap within entomology. This checklist confirms the importance of such documentation of species at both local and regional scale, contributing to knowledge on the region’s biodiversity and its distribution.