The recent increase in the practice of ocular surgery and widespread use of contact lenses have led to an increase in the incidence of severe ocular infections. Inadequate management of these infections can result in irreversible loss of vision. Microbiological diagnosis is essential when the lesions are non-specific, recurrent, or unresponsive to antibiotic therapy, but is hampered by the difficulty of analyzing limited sample volumes containing small inocula. This document presents a review of the most important ocular infections according to the structure affected, and describes the most common causes of these infections and the situations in which a microbiological diagnosis is recommended. Information is included on the sample type and sampling methods, sample transport to the laboratory, and laboratory management and processing techniques, with special attention to liquid culture media. The yield of smear examination and culture of each type of ocular specimen is specified. Lastly, the molecular methods recently developed for the diagnosis of ocular fungal infections and Acanthamoeba infections are described.