Background and objective Mediastinal lymphadenopathy (MLN) in human immunodeficiency virus (HIV) infection has a wide spectrum of aetiologies with different prognoses and treatments. The decision to pursue a histopathological diagnosis represents a clinical challenge as patients present with non-specific symptoms. This study aimed to determine the aetiology and predictive factors of MLN in a cohort of HIV-infected patients in the combination antiretroviral therapy (cART) era. Methods Single-centre retrospective cohort study of 217 consecutive HIV-infected patients who underwent computed tomography (CT) of the chest between January 2004 and December 2009. Fifty-two patients were identified to have MLN (>10 mm in short axis). CT images were re-reviewed by an independent radiologist blinded to the clinical information. Final diagnoses of MLN were obtained from clinical records. Multivariate analysis was performed to identify predictors of aetiology of MLN. Results Seventeen patients (33%) had a diagnosis of malignancy. Consolidation on CT was associated with a reduced likelihood of malignancy odds ratio (OR) 0.03 (95% confidence interval 0.002-0.422), and larger lymph nodes were associated with an increase in the odds of malignancy (OR 2.89; 95% confidence interval 1.24-6.71). CD4 count was found not to be a predictor of aetiology of MLN. Conclusions In the era of combination cART, opportunistic infections and malignancy remain to be the frequent causes of MLN in HIV-positive patients, but the prevalence of non-HIV related malignancy has increased compared with previous studies. Although certain findings are predictors of non-malignant disease, pathological diagnosis of MLN in HIV-positive patients should be pursued whenever possible. See Editorial, page 300 We evaluated radiological, laboratory and clinical data of HIV patients with MLN in the era of combination ART. Opportunistic infections and malignancy remain frequent causes of MLN in HIV-positive patients, but the prevalence of non-HIV-related malignancy has increased compared with previous studies, emphasizing the need for histological diagnosis. © 2014 The Authors. Respirology published by Wiley Publishing Asia Pty Ltd on behalf of Asian Pacific Society of Respirology.