Perforated tympanic membranes (TM) and otitis media can be managed with a paper patch or tympanoplasty. However, a paper patch is not biocompatible and tympanoplasty requires complex aseptic surgical procedures. A novel biocompatible patch with a water-insoluble chitosan as the main component was prepared. Optimal mechanical characteristics of a water-insoluble chitosan patch scaffold (CPS) was approximately 40 microm in thickness, 7 MPa in tensile strength, and 107% in percent elongation, even though the characteristics varied significantly depending on the concentrations of chitosan and glycerol. SEM of the CPSs showed a very smooth surface as compared with that of the paper patches. These CPSs showed no cytotoxicity and had a stimulating effect on the proliferation of TM cells in in vitro study. In in vivo study, 4 (21.1%) and 17 (89.5%) TMs out of 19 adult rats with CPSs showed no perforations at 1 and 2 weeks, respectively. However, left control TMs showed healing of 0 (0%) at 1 week and 18 (94.7%) at 2 weeks. TEM findings of regenerated eardrums using CPSs showed thinner, smoother, and more compact tissues than spontaneously healed eardrums. A CPS was more effective than spontaneous healing to repair traumatic TM perforations.