Sedentary lifestyle and low physical activity are associated with health issues, including both physical and mental health, non-communicable diseases, overweight, obesity and reduced quality of life. This study investigated differences in physical activity and other individual factors among different occupational groups, highlighting the impact of sedentary behaviour on perceived stress by occupation. Cross-sectional study included 571 full-time workers of Kaunas city, Lithuania. The outcome of this study was assessment of perceived stress. Time spent sedentary per day, occupation and other individual characteristics were self-reported using questionnaires. Two main occupational groups were analysed: white-collar and blue-collar workers. Multivariate logistic regression was used to assess the impact of sedentary behaviour on perceived stress among different occupational groups. The prevalence of high sedentary behaviour was 21.7 and 16.8 % among white-collar and blue-collar workers, respectively. Blue-collar workers had a higher risk of high perceived stress (OR 1.55, 95% CI 1.05–2.29) compared to white-collar workers; however, sedentary time did not have any impact on high perceived stress level. Meanwhile, white-collar male (OR 4.34, 95% CI 1.46–12.95) and white-collar female (OR 3.26, 95% CI 1.23–8.65) workers who spend more than three hours per day sedentary had a greater risk of high levels of perceived stress. These findings indicate sedentary behaviour effect on perceived stress among two occupational groups—white-collar and blue-collar workers—and other important factors associated with perceived stress.