Abstract Background Differentiating between unipolar and bipolar depression can be clinically challenging, especially at first presentation. Patterns of cortisol secretion could aid diagnostic discrimination in affective disorders although there has been little comparative research to date. In this study, we investigated acute (saliva) and chronic (hair) cortisol levels concurrently in unmedicated unipolar and bipolar disorders by using conventional diagnostic criteria and self-report measures. Methods Patients with unipolar and bipolar major depression and healthy controls were recruited and assessed. Cortisol levels were extracted from saliva and hair specimens. Depressive features were investigated according to diagnostic groups and with a continuous self-report measure of bipolarity using the Hypomania Checklist (HCL-33). Results Whilst a trend towards a reduction in the total daily salivary cortisol output—area under the curve with respect to the ground (AUCg)—was detected in depressive disorders across diagnosis, the self-administrated bipolarity index suggested that an increase in bipolarity symptoms predicted lower cortisol levels using AUCg. Chronic cortisol measurement did not discriminate unipolar from bipolar depression. Conclusion Results suggested that whilst a low total daily salivary cortisol output (AUCg) might be associated with depressive symptoms, a self-reported measure of bipolarity predicts lower daily cortisol output.