Purpose: This study examined longitudinal changes in health behaviors among Korean female workers according to workplace size using the National Health Insurance Service Female Employee Database (NHIS-FEM DB). Methods: This retrospective longitudinal study used health examination data collected between 2009 and 2015. Female workers aged 15–64 years who completed four health examinations during the study period were included. Propensity score matching based on age and body mass index resulted in 26,787 small- and medium-sized enterprise (SME) workers and 26,787 large enterprise (LE) workers. Smoking, alcohol consumption, and physical activity were compared according to workplace size using descriptive statistics, chi-square tests, and odds ratios (ORs). Results: Current smoking rates decreased in both groups over time; however, LE workers consistently showed higher smoking rates than SME workers. The proportion of workers with smoking duration ≥10 years increased in both groups. Frequent alcohol consumption increased over time, particularly among LE workers, whereas high-volume drinking decreased in both groups. Physical activity levels generally increased during follow-up. Moderate-intensity physical activity showed a greater increase among SME workers than LE workers. Differences in health behaviors according to workplace size persisted throughout the 7-year follow-up period. Conclusion: Health behaviors among Korean female workers changed over time, and workplace size-related differences remained evident during follow-up. These findings suggest that workplace environment and organizational characteristics should be considered when developing health promotion strategies for female workers.