Tsetse flies are the vectors of African trypanosomiasis affecting 36 sub-Saharan countries. Both wild and domestic animals play a crucial role in maintaining the disease-causing parasites (trypanosomes). Thus, the identification of animal reservoirs of trypanosomes is vital for the effective control of African trypanosomiasis. Additionally, the biotic and abiotic factors that drive gut microbiome diversity in tsetse flies are primarily unresolved, especially under natural, field conditions. In this study, we present a comprehensive DNA metabarcoding approach for individual tsetse fly analysis in the identification of mammalian blood meal sources and fly bacterial microbiome composition. We analyzed samples from two endemic foci, Kafue, Zambia collected in June 2017, and Hurungwe, Zimbabwe sampled in April 2014 (pilot study) and detected DNA of various mammals including humans, wild animals, domestic animals and small mammals (rat and bat). The bacterial diversity was relatively similar in flies with different mammalian species DNA, trypanosome infected and uninfected flies, and female and male flies. This study is the first report on bat DNA detection in wild tsetse flies. This study reveals that small mammals such as bats and rats are among the opportunistic blood meal sources for tsetse flies in the wild, and the implication on tsetse biology and ecology needs to be studied.