Background: Happiness is one of the ultimate goals of every human being. Happiness is a significant factor of health system efficiency. Healthcare workers are at the core of every health system. However, up-to-date literature on happiness among healthcare workers is limited. The purpose of this study is to investigate the factors influencing the self-assessed happiness among healthcare workers in public and mission hospitals in Meru County, Kenya. Methods: Using a cross-sectional design, a total of 553 healthcare workers in 24 hospitals completed the Orientations to Happiness questionnaire between June and July 2020. Results: Healthcare workers’ overall happiness was significantly different between hospitals of public and mission ownership (p<0.05). The orientations to happiness mean scores of both pursuits of pleasure and meaning were significantly different between public and mission hospitals (p<0.05). However, there were no statistically significant differences in the pursuit of engagement among the healthcare workers between public and mission hospitals (p<0.05). In both public and mission hospitals, income and the type of toiletry facility were significant factors of overall happiness (p<0.05 or p<0.1). In mission hospitals, eight more variables were statistically significant factors of overall happiness namely type of employment, occurrence of water unavailability, safe drinking water, acceptable main source of water, type of toiletry facility, hospital disposal of garbage, availability of water for hand washing, and overall safety of the hospital working environment (p<0.05 or p<0.1). In public hospitals, additional statistically significant factors of overall happiness were qualification level, and a functional workplace safety and health committee (p<0.05 or p<0.1). Conclusion: Demographic, work-related, and physical work environment factors significantly contribute to healthcare workers overall happiness in both mission and public hospitals. The findings present possible areas of focus for policy and practical implications related to healthcare workers’ happiness aimed at health workforce and health systems strengthening in Kenya.