Mounting evidence connects the biomechanical properties of tissues to the development of eye diseases such as keratoconus, a disease in which the cornea thins and bulges into a conical shape. However, measuring biomechanical changes in vivo with sufficient sensitivity for disease detection has proven challenging. Here, we demonstrate the diagnostic potential of Brillouin light-scattering microscopy, a modality that measures longitudinal mechanical modulus in tissues with high measurement sensitivity and spatial resolution. We have performed a study of 85 human subjects (93 eyes), consisting of 47 healthy volunteers and 38 keratoconus patients at differing stages of disease, ranging from stage I to stage IV. The Brillouin data in vivo reveal increasing biomechanical inhomogeneity in the cornea with keratoconus progression and biomechanical asymmetry between the left and right eyes at the onset of keratoconus. The receiver operating characteristic analysis of the stage-I patient data indicates that mean Brillouin shift of the cone performs better than corneal thickness and maximum curvature respectively. In conjunction with morphological patterns, Brillouin microscopy may add value for diagnosis of keratoconus and potentially for screening subjects at risk of complications prior to laser eye surgeries.