Chronic tympanic membrane (TM) perforation is a common problem worldwide. Recent reports have shown epidermal growth factor (EGF) to stimulate healing in approximately 80% of chronic TM perforations in chinchillas when applied in three doses over 1 week. The objective of this controlled study is to evaluate the efficacy of long-term EGF in the closure of TM perforations. Chronic chinchilla TM perforations were treated with EGF for up to 6 weeks. One hundred percent (17 of 17) of treatment group perforations completely healed. However, two new findings with this long dosing scheme were reperforation on long-term follow-up and three TMs with cholesteatomas. It is likely that reperforation was due to a progressive thinning seen with prolonged EGF application. Long-term EGF use is not recommended for the treatment of TM perforations because of possible wound healing impairment and possible cholesteatoma induction.