Dysfunctions and dystrophies severely affect the cornea’s function. In point of fact, cornea diseases are the second major cause of blindness worldwide. Corneal diagnosis in clinical practice heavily relies on imaging techniques such as slit lamp microscopy, confocal microscopy, or optical coherence tomography. However, these fail to provide information on the cell’s metabolic state or the structural organization of the corneal stroma. With two-photon microscopy and fluorescence lifetime imaging this information can be obtained. Therefore, corneal pathology diagnosis may be improved. The feasibility of corneal characterization by two-photon imaging has been demonstrated in ex vivo samples and in vivo animal models. In this chapter, we report on the use of two multiphoton microscopy instruments for imaging the human cornea: a 5D multiphoton laser scanning microscope and the multiphoton tomograph MPTflex. Human corneas unsuitable for transplantation but otherwise normal and pathological samples obtained after surgery were imaged and characterized based on their autofluorescence and second-harmonic generation signals. Two possible clinical applications of two-photon microscopy are discussed: (i) the assessment of tissue viability before corneal transplantation and (ii) the differential diagnosis of corneal pathologies, further demonstrating the advantages of this imaging modality for corneal diagnosis.