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Fruit flies (Diptera: Tephtritidae) are one of the most important insect pests to fruits worldwide. In Uganda, fruit flies have inflicted considerable yield losses especially in mangos (Mangifera indica L.), However, there has been no recent assessment of the associated economic damage impact despite the outcries from the farming communities. The objective of this study was to assess fruit fly prevalence and infestation in mango and other hosts, to guide the development and improvement of fruit fly control measures in the mango industry in Uganda. A survey was conducted in the major mango producing areas of the country. Four fruit fly (Diptera: Tephritidae) species were recovered from various field-collected fruits; namely Bactrocera invadens, Ceratitis cosyra, Ceratitis rosa and Ceratitis capitata. Bactrocera invadens was the most prevalent species (98%), while C. capitata was the populous. A total of 73% of the mango fruit samples collected from seven agro-ecological zones was found infested with fruit fly larvae. Fruit samples from West Nile had the highest infestation (83%), followed by Northern Moist, Lake Victoria Crescent, and Western Medium High Farmland, with 78, 75 and 73% fruit fly infestation, respectively. It was common to experience 100% fruit losses across the agro-ecological zones in the absence of control measures. The situation was increasingly severest with exotic commercial varieties. Besides mangoes, over 15 other cultivated and wild fruits acted as alternative hosts to the fruit flies.