Currently, about one in five workers is employed in night shift work in Europe. Shift work including nighttime hours is essential in several activities, especially the healthcare sector. Importantly, night working may be associated with the occurrence of sleep disorders or work-related stress, both potentially augmenting the risk of errors and accidents at work. This study aims to examine the presence of neurobehavioral alterations that can be a consequence of shift working and concurrent misalignment of the sleep times and circadian rhythms. Nurses (n = 102) employed at a University Hospital located in North-Eastern Sicily, Italy, voluntarily participated in this pilot study. During medical surveillance, morning and evening salivary samples were collected, and seven psychodiagnostics questionnaires were administered to all the subjects. On one hand, the salivary levels of stress-related biomarkers (cortisol and alpha-amylase) and a circadian biomarker (melatonin) were evaluated. On the other hand, several neurobehavioral features were assessed, including depression, anxiety, work-related, and sleep issues. Interestingly, a positive relationship between salivary morning cortisol and depression scale, as well as a negative relationship between salivary morning alpha-amylase and work ability scale, were observed. Based on these results, the integration of subjective questionnaire outcomes and objective salivary biomarker quantification can help to identify workers with increased susceptibility to developing neurobehavioral alterations. This approach may contribute to ameliorating preventive strategies towards sensitive categories, such as nurses working rotation shifts.