Fruit flies (Diptera: Tephritidae and Lonchaeidae) are among the principal phytophagous insects of fruit and vegetables. Their larvae feed on the pulp or seeds of fruits to complete their development, which favors the entry of pathogens and early fall of the fruits, damaging fruit production. The objectives of this research were to determine the richness of fruit fly species and their associations with their host fruits in the Serra da Bodoquena National Park (Mato Grosso do Sul State, Brazil) and surrounding areas. To obtain the fruit fly species from their hosts, monthly collections of fruits were made. Leaf samples were prepared and identified by botanists. The collected fruits were transported to the Laboratório de Insetos Frugívoros, where they were counted and weighed. The species of fruit flies were identified, and their trophic interactions with their hosts analyzed. Thirty-nine plant species from 29 plant families were collected. Twelve fruit species were hosts to fruit fly larvae. The survey resulted in the recovery of 1,476 larvae and 968 adult fruit flies. Among the adults, 878 were Tephritidae and 90 Lonchaidae. Nine species from the genus Anastrepha (Diptera: Tephritidae) and 3 from Neosilba (Diptera: Lonchaeidae) were obtained. These are the first reports for Anastrepha serpentina (Wiedemann), Anastrepha fraterculus (Wiedemann), and Anastrepha obliqua (Macquart) (all Diptera: Tephritidae) infesting fruits of Eugenia myrcianthes Nied (Myrtaceae); Anastrepha striata Schiner (Diptera: Tephritidae) infesting Jacaratia spinosa Aubl. (Caricaceae); Anastrepha zenildae Zucchi (Diptera: Tephritidae) infesting Campomanesia guazumifolia (Cambess) O. Berg. (Myrtaceae); Neosilba glaberrima (Wiedemann) and Neosilba zadolicha McAlpine & Steyskal are recorded for the first time infesting Persea americana Miller (Lauraceae), and Neosilba certa (Walker) (all Diptera: Lonchaeidae) infesting Chrysophyllum gonocarpum (Mart. & Eichler) Engl. (Sapotaceae), Anastrepha barbiellinii Lima (Diptera: Tephritidae) is recorded for the first time infesting Pereskia aculeata Miller (Cactaceae) in Mato Grosso do Sul State.