Background and Objectives: Despite the importance of nutritional status and a healthy lifestyle in shaping overall well-being, little is known about examining gender-specific differences and trends in health, lifestyle, and nutritional status. The present study aimed to evaluate blood levels of micronutrients, homocysteine, and CoQ10, as well as physical activity (PA) levels and sedentary behavior, among a cohort of Austrian bank staff, with a particular focus on identifying gender differences as well as gender-specific nutritional deficiencies compared to the reference ranges. Materials and Methods: Following a cross-sectional study design, 123 Austrian bank staff (mean age: 43 years; 51% females) participated in this study. Blood samples were collected to evaluate participants’ micronutrient status and serum levels of homocysteine and CoQ10. Whole-blood values of macronutrients were compared to gender-specific reference ranges and categorized into three groups: below, within, or over the range. The WHO’s Global Physical Activity Questionnaire was used to assess PA levels and sedentary behaviors. Results: No significant difference between males and females was found for diet types, PA levels, sedentary time, homocysteine levels, or CoQ10 values (p > 0.05). A high PA level was reported by 64% of males and 58% of females. 71% of females and 56% of males were found to have a vitamin D deficiency. 63–98% of females and 72–97% of males showed normal blood levels for the remaining micronutrients, including potassium, calcium, magnesium, copper, iron, zinc, selenium, manganese, molybdenum, B6, B9, and B12. Conclusions: The findings highlight the necessity of implementing tailored strategies to foster healthy lifestyle behaviors, thereby enhancing the overall state of health, particularly in the context of occupational health.