Retinal nerve fiber layer thickness is affected in several ocular and systemic conditions, most commonly glaucoma and diabetes mellitus. The present cross sectional study was conducted to compare the retinal nerve fiber layer thickness in patients of primary open angle glaucoma and patients of type 2 diabetes mellitus. A total of 120 consecutive eyes of 60 patients were assigned to 2 groups of 30 patients each of primary open angle glaucoma and type 2 diabetes mellitus. Retinal nerve fiber layer thickness was measured with spectral-domain optical coherence tomography. Readings from all the areas of retina (superior nasal, inferior nasal, inferior temporal, superior temporal, nasal upper, nasal lower, temporal lower, temporal upper) were measured in both eyes. Retina nerve fiber layer thickness 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 primary open angle glaucoma and diabetes mellitus. Such patients should not be over treated. The changes in retinal nerve fiber layer thickness can be used to monitor the progression or regression of diseases affecting nerve fiber layer and efficacy of treatment modalities in individual cases.