The formicofauna of temperate Mexican ecosystems have been studied poorly. The objective of this study was to document the number of ant species in the protected natural area of La Malinche National Park, which features a temperate ecosystem in central Mexico. Furthermore, this study serves to update the list of species for Tlaxcala State in this area of the country. During 2015 to 2016, samples were collected along six 400 m linear transects in oak forest and agricultural land (2,700, 2,800, and 2,900 masl), using pitfall traps, baits with either protein or sugar, and hand collection from plants (where ants were foraging for floral nectar, extrafloral nectar, or honeydew). Twenty-three ant species belonging to 14 genera, 11 tribes, and 4 subfamilies were collected in La Malinche National Park. One genus (Brachymyrmex Mayr [Hymenoptera: Formicidae]), 7 species in the park, and 5 species in Tlaxcala State were new collection records. In pitfall traps, 23 species were recorded, where Dorymyrmex insanus Buckley, Formica densiventris Viereck, Lasius mexicanus Wheeler, and Brachymyrmex musculus Forel (all Hymenoptera: Formicidae) were collected exclusively from agricultural land. Temnothorax brevispinosus MacKay and Stenamma huachucanum Smith (both Hymenoptera: Formicidae) were collected exclusively in the oak forest, whereas 17 species were recovered from both habitats. Nine species were recorded from either protein or sugar baits; Formica propatula Francoeur and Temnothorax texanus Wheeler (both Hymenoptera: Formicidae) were recorded only in tuna baits, and with the remainder of the species collected with tuna and honey. Seven ant species were recorded from hand collections on plants. We have increased the number of ant species in Tlaxcala State from 53 to 58 (28 of which are present in La Malinche National Park). This faunal survey demonstrated that there is a great diversity of ant species in this temperate ecosystem. It is urgent to document the diversity of formicofauna in these types of ecosystems because they may possibly be the first to be negatively affected by climate change.