Retinal nerve fiber layer thickness is affected in several ocular and systemic conditions, most commonly glaucoma and diabetic retinopathy. 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 primary angle glaucoma suffering from 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 patients of primary open angle glaucoma having 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. The presence of type 2 diabetes mellitus in patients of primary open angle glaucoma significantly affected the thickness of retinal nerve fiber layer specially in superonasal and inferotemporal quadrants as compared to patients of primary open angle glaucoma. Retinal nerve fiber layer thickness is 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 having diabetes mellitus, and 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.