Background and Objectives: Health professionals’ working schedules often do not allow them to follow a balanced way of life, and the nature of their work can affect their weight and eating habits. The aim of the present study is to shed some light on the impact of night shift working on the dietary practices of healthcare practitioners in Greece. Materials and Methods: This was a cross-sectional study. With the use of an administered questionnaire several parameters were assessed, including anthropometric characteristics, weight history, as well as nutritional habits of the same group of participants during night shifts and when at regular working hours. Moreover, nutritional beliefs of the subjects were recorded, as well as the level of adherence to the Mediterranean diet. Results: It was observed that in all food groups, including water, used to compare the nutritional patterns of the participants during night shifts and when working regular hours, statistical differences were noted (p = 0.001). Furthermore, the participants who reported a higher adherence to the Mediterranean diet and self-reported healthier nutritional habits, had a statistically lower weight compared to the participants that rarely followed the specific model or reported unhealthier eating routine (p = 0.010 and p = 0.015, respectively). Conclusions: We conclude that shift-working healthcare professionals are associated with disturbed eating behaviours leading to the high consumption of unhealthy food. The implementation of interventions that will concentrate on educating health workers on balanced nutrition and improving physical environment (i.e., food availability, healthier food choices) seems to be of great importance.