Chronic alcohol use is a common problem globally among the HIV-infected patients on ARV treatment regimens, leading to severe liver damage and increase in serum enzymes. The study determined effect of chronic alcohol intake on serum enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST) and ?-glutamyl transferase (GGT)) in HIV-infected patients on d4T/3TC/NVP treatment regimen in Uganda using the WHO alcohol use disorders’ identification test (AUDIT) tool and chronic alcohol use biomarkers (ALT, AST, GGT, AST/ALT = 2.0 and mean corpuscular volume (MCV)). A case control study using repeated measure design with serial measurements model was used. Alcohol use biomarkers were used to standardize the gender differences in alcohol use. A total of 41 patients (21 alcohol group and 20 control group) were followed up for 9 months with blood sampling done at 3 month intervals. The serum enzymes’ levels were determined by using the Cobas Intergra 400 Plus analyzer system. The mean GGT levels were higher in chronic alcohol use group as compared to control group in both groups. The levels were above reference ranges during 6 month and three times higher during 9-month follow-up period for both chronic alcohol use self reporting WHO AUDIT tool and biomarkers’ groups. Generally, the mean AST, ALT and AST/ALT levels were slightly higher in alcohol use group as compared to control group and were slightly higher in both groups as compared to reference ranges during the 9 month follow-up period. Chronic alcohol consumption by HIV-infected patients on d4T/3TC/NVP drug regimen increased GGT and AST/ALT serum enzyme levels and hence was used as chronic alcohol use biomarkers.