The Neotropical genus Mesoconius Enderlein, 1922 is revised with the redescription or diagnosis of 20 previously described South American species, the description of one new species of the M. infestus group (M. triunfo sp. nov.) from Mexico and the description of 32 new species in four species groups from Andean South America (M. eques group: M. albiseta sp. nov., M. albitergum sp. nov., M. anchitarsus sp. nov., M. cosanga sp. nov., M. epandribarba sp. nov., M. hirsutimamma sp. nov., M. nigripleuron sp. nov., M. noteques sp. nov., M. pasachoa sp. nov., M. rufipleuron sp. nov. and M. suzukii sp. nov.; M. infestus group: M. acca sp. nov., M. albipedis sp. nov., M. nigricephala sp. nov. and M. notacca sp. nov.; M. nono group: M. aurantium sp. nov., M. bipleuron sp. nov., M. garyi sp. nov., M. nono sp. nov., M. reinai sp. nov., M. uchumachi sp. nov., M. woytkowskii sp. nov. and M. zorro sp. nov.; M. oblitus group: M. apa sp. nov., M. apicalis sp. nov., M. gelbifacies sp. nov., M. keili sp. nov., M. lobopoda sp. nov., M. nigra sp. nov., M. quadritheca sp. nov., M. rex sp. nov. and M. ruficrus sp. nov.). Calobata eques Schiner, 1868 is transferred from Cliobata Enderlein, 1923 to Mesoconius and all South American species previously treated as Zelatractodes Enderlein, 1922 are transferred to Mesoconius. Mesoconius aeripennis Enderlein, 1922 is synonymized with M. eques, M. enderleini Frey, 1927 is synonymized with M. infestus Enderlein, 1922 and Aristobata melini Frey, 1927 is synonymized with M. filipes (Enderlein, 1922). Mesoconius garleppi (Enderlein, 1922) is newly recognized as a subjective junior homonym and given the replacement name Mesoconius ottoi nom. nov. A maximum likelihood tree is provided for 29 species of Mesoconius sequenced for the barcode region of CO1 and a key is provided for all South American species of Mesoconius.