In difficult or refractory cases, the ideal management of intraocular inflammation and malignancy may require a tissue diagnosis. Diagnostic vitrectomy is one well-established method for acquiring such tissue; however, in some patients, the diagnostic pathology is limited to the choroids and retina, making vitrectomy alone insufficient. In such cases, full-thickness retinal and/or choroidal biopsies of the involved tissues may be useful. Such ocular biopsies may be analyzed by routine and molecular pathology, immunohistochemistry, immunology and microbiology. Prompt handling and adequately processing specimens is invaluable in increasing the yield of these procedures. The utility of ocular biopsy in diagnosis of ocular inflammatory, infectious and malignant diseases are illustrated and summarized in several classical reported cases. With careful consideration of clinical manifestations, ocular biopsies and tissue sampling will help to improve and confirm clinical diagnosis, understanding of disease pathogenesis, and guide appropriate therapy.