The data set includes the results of a laboratory analysis in 2016, investigating the presence of trypanosomes and prevalence of tsetse endosymbionts in tsetse flies. The tsetse flies were sampled in Hurungwe District, Mashonaland West Province, Zimbabwe, from February 2014 to November 2014. Flies were sampled using a combination of Epsilon traps and fly rounds, both established techniques for sampling tsetse. Tsetse were stored prior to laboratory analysis using Polymerase chain reaction (PCR) techniques in 2016. The data include two species of tsetse, Glossina pallidipes and Glossina morsitans morsitans. Trypanosome species investigated include Trypanosoma brucei s.l., T. b. rhodesiense, T. vivax, T. congolense, T. simiae, T.simiae (Tsavo) and T. godfreyi. Endosymbionts included in the study were Sodalis glossinidius and Wolbachia spp. Hurungwe District is the only sleeping sickness focus in Zimbabwe and an increase in cases had been detected in years preceding this study. The objective of the study was to investigate the trypanosome species present in the tsetse population and their association with tsetse endosymbionts. This study was conducted as part of research into the relationship between trypanosomiasis, well-being and ecosystems by the Dynamic Drivers of Disease in Africa Consortium (DDDAC). The research was funded by NERC with support from the Ecosystem Services for Poverty Alleviation Programme (ESPA). Full details about this dataset can be found at https://doi.org/10.5285/1e435dd3-ed93-4961-a136-ffa477731e1c