Background The widespread problem of burnout among healthcare workers is not only common but also a significant concern that impacts the entire healthcare system in Uganda. It is essential to understand the connection between burnout and quality of life among healthcare workers in the specific context of central Uganda, where healthcare professionals face high patient volumes, limited resources, exposure to infectious diseases, and socioeconomic challenges. This study examined the relationship between burnout and quality of life among healthcare workers in central Uganda. Methods This research utilized a cross-sectional study conducted across various healthcare settings in central Uganda. The data were analyzed at descriptive, bivariate, and multivariate levels. The relationship between dependent and independent variables was evaluated using an independent t-test for binary variables and a one-way analysis of variance (ANOVA) for categorical variables. Significance was determined with a reported p-value, with relationships deemed significant at p < 0.2. For multivariable analysis, multiple linear regression was employed using a forward selection method, with significance set at 5% (p < 0.05). Results Our findings indicate that nearly 40% of healthcare workers reported experiencing high levels of burnout. The average score for overall quality of life was 10.71 (±4.89), with variations observed across different domains. The study reveals a significant connection between socio-demographic factors, burnout, and overall quality of life, emphasizing the impact of job category, supervisory support, sleep quality, and burnout on the well-being of healthcare workers. Predictive analysis illustrates how these factors influence both overall quality of life scores and scores in specific domains. Particularly noteworthy is that nurses and technicians tend to have a lower quality of life compared to physicians. Conclusion The results underscore the relationship between socio-demographic factors, burnout, and particular aspects of quality of life. Notably, job category, supervisory support, sleep quality, and burnout stand out as significant factors shaping the well-being of healthcare workers. Nurses and technicians encounter distinct challenges, suggesting the need for interventions tailored to their needs. Addressing issues such as inadequate supervisory support, burnout, and sleep-related problems is recognized as a potential approach to improving the overall quality of life among healthcare workers.