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Abstract Background: Animal diseases that are endemic like tsetse transmitted trypanosomosis cause the continuous expenditure of financial resources of livestock farmers and loss of productivity of livestock. Estimating the cost of controlling animal trypanosomosis can provide evidence for priority setting and targeting cost-effective control strategies. Methodology: A cross-sectional survey to estimate the economic cost of bovine trypanosomosis was conducted in cattle-keeping communities living around Murchision falls National Park, in Buliisa district Uganda. Data was collected on herd structure, the cost of treatment and control, prevalence of morbidity and mortality rates due to trypanosomosis, and salvage sales losses in cattle herds in the last year. Results: In this study, 55.4% (n = 87) of the households reported their cattle had been affected by trypanosomosis during the previous last year. There was a high economic cost of trypanosomosis (USD 653) per household in cattle-keeping communities in Buliisa district of which 83% and 9% were due to mortality and milk loss respectively/ High mortality loss was due to low investment in treatment. The study showed that prophylactic treatment 3 times a year of the whole herd of cattle using Samorin ® (Isometamidium chloride) at a cost of USD 110 could drastically reduce cattle mortality loss due to trypanosomosis due to trypanosomosis with a return on investment of USD 540 annually per herd. This could be coupled with strategic restricted insecticide spraying of cattle with deltamethrin products. Conclusion: The results show a high economic cost of trypanosomosis in cattle-keeping communities in Buliisa district, with cattle mortality contributing the largest proportion of the economic cost. The high mortality loss was due to low investment in treatment of sick cattle.