Many artificial materials have been used for mastoid cavity obliteration in canal wall down tympanomastoidectomy. Among the artificial materials, hydroxyapatite is most popular material for mastoid cavity obliteration. Hydroxyapatite is biostable and osteoconductive, but it has poor mechanical properties, especially brittleness. To overcome the limitation, hydroxyapatite has been combined with natural chitosan polymer, which is biocompatible and flexible, to produce a hydroxyapatite-chitosan patch via a sublimation-assisted compression process. We evaluated this hydroxyapatite-chitosan patch for tympanic cavity obliteration in a rat model by comparing it with sham surgery group, bone cement and homologous cartilage. The cavity obliteration ratio and new bone formation in tympanic bullae were determined by computed tomography, histological analysis, field emission scanning electron microscopy, and energy-dispersive X-ray spectroscopy. Comparing the hydroxyapatite-chitosan patch with homologous cartilage and bone cement, the tympanic cavity obliteration ratio was superior in the patch group (40.96 ± 7.79%) than other groups (32.23 ± 6.93% and 16.07 ± 6.10%). Even the hydroxyapatite-chitosan patch made no difference in new bone formation ratio ranging from 27.92 ± 11.08 to 29.17 ± 10.96% with other groups. In conclusion, hydroxyapatite-chitosan patch for mastoid cavity obliteration showed easy handling, flexibility, favorable osteogenesis and biostability.