The purpose of this study was to investigate the personal, interpersonal, and organizational factors associated with cardiovascular disease (CVD) prevention behavior among office workers using an ecological model. A total of 221 office workers working in areas such as personnel, design, and accounting, and who work in offices, completed the survey. Health Perception Questionnaire, General Self-Efficacy Scale (GSE), Multidimensional Scale of Perceived Social Support (MSPSS) Scale, the Korean Occupational Stress Scale–Short Form (KOSS-SF), and Social Environment Influencing Health form were used to measure each factor. Health perception (β = .207, p = .003), family support (β = .185, p = .005), and organizational health culture (β = .175, p = .012) were found to have a significant association with the CVD prevention behaviors of office workers. Comprehensive and multidimensional intervention is essential to enhance the CVD prevention behaviors of office workers. This study informs the development of a workplace-based intervention program to prevent CVD among office workers.