The dataset describes the results of a laboratory analysis investigating the presence of various infectious agents in goats, cattle, pigs, dogs and sheep from Mambwe District, Eastern Province, Zambia. Blood samples were collected in June, July and August 2013 and stored on Whatman FTA (Flinders Technology Associates) cards. Laboratory analysis was conducted using polymerase chain reactions (PCR) for African trypanosomes and tick-borne infections. In addition, serum was tested for Brucella using the Rose Bengal test. Cattle and dogs were tested for African trypanosomes, tick-borne infections and Brucella. Goats and sheep were tested for African trypanosomes and Brucella. Pigs were tested for African trypanosomes only. The objective was to evaluate the health status of domestic animals in the Mambwe District. This work was conducted alongside a human wellbeing questionnaire survey. The research was part of a wider research project, the Dynamic Drivers of Disease in Africa Consortium (DDDAC). The research was funded by NERC project no NE/J000701/1 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/f81ede76-a1d4-4367-aa8c-de087350457e