Objective: To compare the diagnostic rate between in vivo confocal microscopy and pathological examination in retrocorneal fungal infection. Methods: It is a retrospective study. A total of 18 patients with retrocorneal fungal infection and received PKP surgery in the Qingdao Eye Hospital from November 2009 to December 2020 were enrolled. KOH smear and in vivo confocal microscopy examination were performed before surgery, and pathological examination including periodic acid-schiff (PAS) stain and Grocott Methenamine Silver (GMS) stain were performed after surgery. Patients were diagnosed retrocorneal fungal infection based on in vivo confocal microscopy and pathological examination. The diagnostic rates of the two methods were compared. Results: None of the 18 patients with posterior corneal fungal infection were found to have fungal hyphae in the corneal smear.Sixteen patients (88.9%) were found fungal hyphae by in vivo confocal microscopy. Corneal stroma necrosis and a large number of inflammatory cells were shown by postoperative pathologic examination, and all patients were found fungal hyphae in posterior corneal stroma with PAS stain and GMS stain. Conclusion: Confocal microscopy has unique advantages such as non-invasive and rapid examination in the diagnosis of fungal keratitis.However, it needs to combine with pathological examination for diagnosing the retrocorneal fungal infection.