Human immunodeficiency virus (HIV) infection contributes to both neurological and psychiatric disorders. People with HIV (PWH) are disproportionately affected by chronic pain and negative affective comorbidities, such as generalized anxiety disorder (GAD), major depressive disorder (MDD), and post-traumatic stress disorder (PTSD). PWH are also more likely to consume alcohol, further exacerbating these conditions. Dysregulation of biological stress systems, including the hypothalamic-pituitary-adrenal (HPA) axis, is believed to contribute to these comorbidities. The current analysis sourced data from the New Orleans Alcohol Use in HIV (NOAH) study, a longitudinal investigation of alcohol use patterns in an underserved cohort of PWH. Prior findings from this cohort demonstrated that higher alcohol consumption is associated with worse mental health outcomes (e.g., greater depression and anxiety). Here we examined the relationships between negative affective comorbidities (anxiety, depression, and PTSD), pain, alcohol use, and plasma levels of cortisol, the major stress hormone, in PWH. Our analysis revealed significant associations between cortisol levels, PTSD symptoms, and pain intensity. Moreover, the relationship between PTSD and cortisol was stronger among recent alcohol drinkers (PEth-positive participants) and males. The association between pain intensity and cortisol was also stronger in recent alcohol drinkers. Although depression and cortisol levels showed no overall relationship, females with the highest depression scores exhibited significantly higher cortisol levels. Our findings emphasize the need for further investigation into how ongoing alcohol use may increase relationships between cortisol and the deterioration of mental health in male and female PWH.