Retinal nerve fiber layer thickness (RNFLT) is affected in several ocular and systemic conditions, most commonly glaucoma and diabetic retinopathy. This cross sectional study was done to compare the RNFLT in 180 eyes of 90 patients. A total of 90 patients were assigned to 3 groups of 30 patients each of primary open angle glaucoma (POAG), type 2 diabetes mellitus (T2DM) and POAG with T2DM (POAG/T2DM). The RNFLT was measured with spectral-domain OCT. Readings from all the areas of retina were measured in both eyes. Presence of T2DM in patients of POAG significantly affected the RNFLT as compared to patients of POAG or T2DM individually. The RNFLT was negatively correlated with the duration of glaucoma, duration of diabetes and HBA1c levels. Hence, care should be taken in interpreting optical coherence tomography readings in patients of POAG/T2 DM. The changes in RNFLT can be used to monitor the progression of diseases affecting RNFLT and efficacy of treatment modalities.