Although nurses’ fatigue affects their well-being and patient safety, no effective objective measurements exist. We explored the profiles of salivary biomarkers associated with nurses’ chronic fatigue across several shifts. This longitudinal study involved 45 shiftwork nurses and collected their saliva samples before two night and two day shifts for a month. Chronic fatigue was measured using the Cumulative Fatigue Symptom Index before the first night shift. Biomarker profiles were analyzed using hierarchical cluster analysis, and chronic fatigue levels were compared between the profiles. Cortisol profiles were classified into high- and low-level groups across two day shifts; the low-level group presented significantly higher irritability and unwillingness to work. Secretory immunoglobulin A (s-IgA) profiles across the four shifts were classified into high- and low-level groups; the high-level group had significantly higher depressive feelings, decreased vitality, irritability, and unwillingness to work. Cortisol (two day shifts) and s-IgA (four shifts) profiles were combined, and (i) cortisol low-level and s-IgA high-level and (ii) cortisol high-level and s-IgA low-level groups were identified. The former group had significantly higher chronic fatigue sign and irritability than the latter group. The profiles of salivary cortisol and s-IgA across several shifts were associated with nurses’ chronic fatigue.