The VALIDATE Network brings together scientists addressing vaccine development for neglected infectious diseases caused by intracellular pathogens. In September 2023, the first workshop on One Health approaches to research and capacity building was held in Paarl, South Africa, with a focus on tuberculosis and leishmaniasis. Thirty-two scientists from 15 countries presented and discussed broad topics pertinent to zoonotic diseases, cross-species disease transmission, disease mitigation strategies, inequitable access to medicines and health technologies, and health system challenges in One Health. In this report, we summarize the gaps, challenges, and opportunities identified during the 2023 VALIDATE One Health workshop. We anticipate that the experiences and dialogues will be informative for animal, human, and environmental health investigators to guide the development of research projects and vaccine development with a One Health vision.