The Central America collection contains sets of pottery vessels and stone sculptures from Ancient Mesoamerica (Monte Alban, Teotihuacan, Veracruz, Aztecs, Mayas) and a numerous collection of pottery from Chiriqui in Panama. The notable figures who contributed to this collection were Jakub Beer, Grandmaster of the Knights of the Cross with the Red Star, who in 1856 donated a large Central American collection to Bohemia, and the French-Mexican amateur archaeologist and collector August Genin, who out of respect for president T. G. Masaryk donated a collection of pre-Columbian art to Czechoslovakia, in particular Colima, Zapotek and Aztec ceramics. The modern history acquisitions include ethnographic items of the present-day indigenous Nahua, Maya, Mixtek, Zapotek and Huichol peoples, and the items of mestizo culture (textiles, masks, ceramics, toys, ritual objects).

The South American collection consists of archaeological and ethnographic material, with colonial art forming an important part of it. The archaeological collections document Ancient Ecuador, Columbia, Venezuela and Peru (La Tolita, Manabi, Bahia, Tiwanaku, Nazca, Moche, Chimú, Chancay and Inka cultures). Scientifically valuable ethnographic items were collected primarily in tropical South America – the core of the ethnographic collection is made up of the sets gathered by the traveller E. St. Vráz in the late 19th century in the basins of the Orinoco and Amazon rivers and by A. V. Frič in the early 20th century in the Gran Chaco region. There are two other sets of artefacts that are of great importance. The first was gathered by the director of the Náprstek Museum, V. Šolc, from the Aymara and Mapuche peoples of Bolivia and Chile in the 1970s and 1980s, and the second is a comprehensive collection relating to Mapuche culture of central Chile, gathered by the anthropologist M. Stuchlík during his field research in the 1960s and the 1970s.