Ocular fungal infections (OFIs) include a variety of conditions such as keratitis, endophthalmitis (both endogenous and exogenous), blepharitis, blepharoconjunctivitis, dacryocystitis, choroiditis, and orbital cellulitis. Accurate laboratory diagnosis of OFIs relies on several factors, including the anatomical site of the infection, the specific type of fungal involvement, and the correct collection, handling, and transportation of clinical specimens. Various laboratory techniques are employed to diagnose these conditions, depending on the clinical presentation. Direct microscopic examination with potassium hydroxide (KOH) is frequently used to detect fungal elements, revealing characteristic structures such as hyaline, pigmented, or yeast-like hyphae. In cases involving dimorphic fungi, distinctive morphological features can often be identified. Additional staining methods—such as Giemsa, Wright, and Periodic Acid-Schiff (PAS)—are commonly used to enhance the visualization of fungal structures. Fungal cultures on Sabouraud dextrose agar, with and without the addition of antibiotics, remain the gold standard for definitive identification of ocular fungal pathogens. Micromorphological evaluation, combined with molecular methods such as polymerase chain reaction (PCR) and proteomic approaches like MALDI-TOF mass spectrometry, enables both presumptive and species-level identification. In vitro antifungal susceptibility testing is routinely conducted to guide appropriate therapeutic strategies. In vivo confocal microscopy serves as a valuable, noninvasive diagnostic tool and should be integrated into the overall diagnostic workflow. Although histopathological examination can confirm fungal infection, it requires invasive sampling procedures and may result in considerable patient discomfort. This chapter reviews the current methodologies employed in the laboratory diagnosis of ocular fungal infections.