Structural disorder of the temporomandibular joint (TMJ) is a progressive disease with poor prognosis due to its physiological three-dimensional anatomical position and the complicated relationship among muscles, ligaments, and cartilage. The lack of detection methods for changes in the collagen structure of the TMJ disc makes the diagnosis untimely and unclear. This work aimed to explore the feasibility of using a promising detection technique, second-harmonic generation (SHG), to characterize collagen fibers in a TMJ disc with structural disorders. The TMJ discs with structural disorder were observed using SHG microscopy, and assessment of collagen orientation was conducted by analyzing digitized images. The SHG images were also compared with the scanning electron microscopy images and microscopic images acquired after hematoxylin and eosin and Masson's trichrome staining. The SHG imaging showed that the collagen fibers in diseased TMJ were distributed in a disorderly manner, and pixel intensities in diseased TMJ discs were significantly different from those acquired in healthy TMJs. Moreover, the three dimensions of collagen fibers and dynamic images acquired by SHG nonlinear optical microscopy showed the structural disorder of the collagen fibers in a diseased TMJ. In summary, SHG imaging could provide three-dimensional and quantitative data, with dynamic and critical pathological information for clinical diagnosis, showing its potential value in the diagnosis and evaluation of structural disorders of the TMJ disc.