The Neotropical family Mesembrinellidae is revised. A total of 53 valid, extant species are included in the family, including 15 described as new and 38 redescribed based on study of type and non-type material and of the literature. A total of 18 primary types were examined. An additional ca. 2300 specimens, belonging to 47 species, were studied in detail, including dissection and photographic documentation of terminalia, with many females illustrated for the first time. Keys to subfamilies, genera, species-groups and species are provided. Type specimens of six species housed in South American institutions could not be obtained for study, i.e., M. bequaerti Séguy, 1925 and the five recently described species M. andina (Wolff et al., 2014), M. carvalhoi (Wolff et al., 2013b), M. cordillera (Wolff & Ramos-Pastrana in Wolff et al., 2017), M. obscura (Wolff in Wolff et al., 2017) and Laneella patriciae (Wolff, 2013). We accept the synonymy, proposed by previous authors, of Eumesembrinella Townsend, 1931 with Mesembrinella Giglio-Tos, 1893. In addition, we synonymize the genera Albuquerquea Mello, 1967, Giovanella Bonatto in Bonatto & Marinoni, 2005, Henriquella Bonatto in Bonatto & Marinoni, 2005, Huascaromusca Townsend, 1918 and Thompsoniella Guimarães, 1977 with Mesembrinella Giglio-Tos, 1893, synn. nov., retaining three valid genera in the family: Laneella Mello, 1967, Mesembrinella and Souzalopesiella Guimarães, 1977. Laneella nigripes Guimarães, 1977 and Mesembrinella bellardiana Aldrich, 1922 are fixed as the type species of the genera Laneella Mello, 1967 and Mesembrinella Giglio-Tos, 1893, respectively, under Article 70.3 of the ICZN Code. We separate Mesembrinella into the following species-groups: M. latifrons (Mello, 1967), M. spicata Aldrich, 1925, M. bolivar (Bonatto in Bonatto & Marinoni, 2005), M. aeneiventris (Wiedemann, 1830), M. bicolor (Fabricius, 1805), and M. anomala (Guimarães, 1977). The following 15 new species are described: Laneella fusconitida Whitworth, sp. nov. from Costa Rica, Ecuador and Venezuela, Laneella fuscosquamata Whitworth, sp. nov. from Guatemala and Mexico, Laneella purpurea Whitworth, sp. nov. from Costa Rica, Mesembrinella bullata Whitworth, sp. nov. from Bolivia, Mesembrinella chantryi Whitworth, sp. nov. from French Guiana and Brazil, Mesembrinella epandrioaurantia Whitworth, sp. nov. from Venezuela, Mesembrinella guaramacalensis Whitworth, sp. nov. from Venezuela, Mesembrinella longicercus Whitworth, sp. nov. from Bolivia, Mesembrinella mexicana Whitworth, sp. nov. from Mexico, Mesembrinella nigrocoerulea Whitworth, sp. nov. from Costa Rica, Ecuador and Venezuela, Mesembrinella serrata Whitworth, sp. nov. from Peru, Mesembrinella velasquezae Whitworth, sp. nov. from Venezuela, Mesembrinella violacea Whitworth, sp. nov. from Costa Rica, Mesembrinella woodorum Whitworth, sp. nov. from Ecuador, and Mesembrinella zurquiensis Whitworth, sp. nov. from Costa Rica. Mesembrinella abaca Hall, 1948 is proposed as a junior synonym of Mesembrinella socors (Walker, 1861), syn. nov. Lectotypes are designated for Dexia randa Walker, 1849 (now Mesembrinella) and Mesembrinella pictipennis Aldrich, 1922. We analyze the most extensive DNA-barcode dataset for Mesembrinellidae to date, encompassing the three genera considered valid and including 188 sequences (178 new) from 35 species, with data for 23 species provided for the first time. The topology of the resulting Neighbor-Joining tree is mostly congruent with morphology; however, some species show considerable genetic variation that is not reflected by morphology. Finally, we include a corrigendum to the recent Zootaxa paper on Nearctic Calliphora Robineau-Desvoidy (Diptera: Calliphoridae) by Tantawi et al.