Work organization, such as shifts and night work, can interfere with the perception of work-related stress and therefore on the development of pathological conditions. Night shift work, particularly, can have a negative impact on workers’ wellbeing by interfering with the biological sphere. The aim of this study is to evaluate the associations between work activities, shift work effects and stress-related responses in 106 dock workers enrolled in southeast Italy. Dock workers’ tasks consist of complex activities that seemed to affect more sleep quality than work-related stress. An analysis of salivary biomarkers such as cortisol, α-amylase, melatonin and lysozyme was performed along with validated psycho-diagnostic questionnaires. Alpha-amylase showed a significant negative correlation with the effort/reward imbalance score; thus, the measurement of salivary α-amylase is proposed as a sensitive and non-invasive biomarker of work-related stress. This study may provide new insights into developing strategies for the management of night shift work. Salivary biomarkers should be further investigated in the future in order to develop simple and effective tools for the early diagnosis of work-related stress or its outcomes.