The Chaco is an international biome, connecting four countries: Paraguay (230,000 km2), Bolivia (90,000 km2), Argentina (520,000 km2), and Brazil (Mato Grosso do Sul state (MS), with around 9,000 km2 and in the middle of South America. Brazilian Chaco is restricted to Porto Murtinho region, MS. The daisies (Asteraceae) with near 24,000 species worldwide is characterized by herbs and shrubs that coevolved with several taxa of endophagous insects: dipterans Agromyzidae, Ceciidomyidae and Tephritidae; Coleoptera (Apionidae), Hemiptera (Miridae), Lepidoptera (Blastobasidae, Gelechiidae, Pterophoridae, Pyralidae, and Tortricidae) and the parasitoids of this endophagous insects, which found in the daisies’s flower heads ideal conditions for food, breeding site and shelter. The Neotropical florivorous flies are the Agromyzinae (Agromyzidae), and Tephritinae (Tephritidae), which in their larval stage feed on Asteraceae inflorescences. To report the species of florivore flies, their host plants and parasitoids in flower heads of Asteraceae from the Brazilian Chaco, we sampled inflorescences of 25 species (± 500 flower heads/species) that were kept in containers to the emergence of the florivorous flies or their parasitoids sampled in the three phytophysiognomies. The adult insects after 48 hours of their emergence were fixed in 80% ethanol for later identification. A total 25 species of Asteraceae were evaluated in the Brazilian Chaco, being collected 17,000 flower heads. Nine tribes of two Asteraceae subfamilies were sampled, from which 15 species of florivorous flies were recovered. We found 5 genera with 9 of Tephritinae (Tephritidae), 6 species of Melanagromyza (Agromyzinae, Agromyzidae), and 104 parasitoids (Hymenoptera) of the florivorous flies.