Nucleoside analogue reverse transcriptase inhibitor (NRTI)-associated mitochondrial toxicity is an important issue in the clinical management of HIV infection. The aim of this study was the detection of mitochondrial dysfunction by flow cytometry in lymphocytes from HIV-infected individuals and its association with blood lactate levels, clinical and virologic status, and the different NRTI-based therapies. Lower peripheral blood lymphocytes with mitochondrial dysfunction (PBLmd) percentages were observed in healthy controls (1.2, interquartile range [IQR] = 0.4–1.9) than in patients (2.2, IQR = 0.9–3.7; P < 0.01). Stavudine-containing therapy showed higher PBLmd percentages (3.0, IQR = 1.1–4.5) than no treatment (2.1, IQR = 0.8–2.8; P < 0.05) or zidovudine-based therapy (0.9, IQR = 0.3–1.4; P < 0.01). A significant inverse correlation was found between PBLmd and CD4 T-cell percentage and absolute count. Patients with an AIDS diagnosis had higher PBLmd percentage (2.7, IQR = 1.1–4.4) than HIV-positive non-AIDS patients (1.4, IQR = 0.6–3.0; P = 0.012). In multivariate analysis, use of stavudine (odds ratio [OR] = 5.86, 95% CI = 1.81–19.01, P = 0.003) and CD4 T-cell counts <200/μL (OR = 4.51, 95% CI = 1.38–14.70, P = 0.012) were independent predictors of high PBLmd percentage. This cross-sectional study shows that antiretroviral drugs can impair the in vivo mitochondrial function of PBLs.