The analysis of the impact of shift work on occupational health still needs further contributions. Therefore, we developed this research with the purpose of assessing the impact of shift work on occupational health indicators, namely burnout, work-engagement, occupational self-efficacy, and mental health functioning (symptoms of depression and anxiety), by comparing workers who did shift work (44.2% of participants) with workers who did not (55.8% of participants). A total of 695 Portuguese professionally active adults between 18 and 73 years of age (Mage = 37.71; SD = 12.64) participated in this study and completed a survey containing a sociodemographic questionnaire and four occupational health measures: The Burnout Assessment Tool, The Work-Engagement questionnaire (UWES), The Occupational Self-Efficacy Questionnaire, and the BSI-18 for mental health symptoms. Results showed statistically significant differences (p < 0.05) for all indicators, demonstrating that participants who worked shifts presented lower scores of work-engagement and occupational self-efficacy, and higher scores of burnout, depression, and anxiety when compared to participants who did not work shifts. Linear regressions showed that shift work explained significant but low percentages of anxiety symptoms, low work-engagement, depression symptoms, low occupational self-efficacy, and burnout. We concluded that non-standard working hours (by shifts) are detrimental to employee occupational health, by increasing the risk of anxiety and depression levels, and burnout, and by reducing work-engagement (as a well-being indicator) and occupational self-efficacy perceptions.