BackgroundThe One Health (OH) approach integrates multiple competencies in the prevention and control of disease outbreaks. Through a range of OH competence-based activities, the Africa One Health University Network (AFROHUN) built the capacity of selected students at Makerere University and Mbarara University of Science and Technology. This study applied the Systems Theoretical Framework (STF) of career development to establish the employment status of AFROHUN-Uganda alumni, and the facilitators and barriers for application of the OH approach in their organisations.MethodsWe conducted a cross-sectional study using mixed methods to collect and analyse data from a random sample of 182 OH alumni of the 2013-2018 cohorts. For quantitative data, descriptive statistics were computed using Stata 14.0 statistical software. A total of 12 in-depth interviews were conducted, and NVivo 12 Pro was used to organise data during thematic analysis.ResultsWhile the majority, 87.4% were or got employed after participating in the AFROHUN Uganda capacity building programme, 68.1% were employed at the time of the survey, 57.7% have worked with their current employer for at least a year, and 39% hold managerial positions. The facilitators of applying the OH approach into employing organisations included being knowledgeable about OH, the presence of a multidisciplinary workforce, the nature of activities implemented, and existing partnerships and collaborations between organisations. The barriers to the application of the OH approach included limited funding, a negative attitude towards working with people from other disciplines, and limited knowledge of the One Health approach.ConclusionNotably, more than two-thirds of the OH alumni are employed, and more than a third hold managerial position. While these findings portray a fairly good absorption rate of the OH alumni into the workforce, they also highlight the facilitators of application of the OH approach that need to be promoted as well as the barriers that need to be addressed if the application of the OH approach is to be improved within the workforce.