Abstract Introduction Cholesteatoma is a disease with significant clinical impact but is incompletely understood. The challenge of performing studies with long-term follow-up in humans is a factor that has restricted the advance of knowledge in this field. Thus, the use of animal models is highly pertinent, and the Mongolian gerbil model has emerged as one of the most useful. Objective The present study aims to evaluate, through serial otoendoscopies, the development and characteristics of pars flaccida retraction pocket and cholesteatoma in Mongolian gerbils after obliteration of the eustachian tube and compare it with the control group. Methods Forty Mongolian gerbils were divided into two groups of 20 animals each. In the intervention group, the animals were followed with serial otoendoscopies after eustachian tube obliteration. In the control group, the animals were only followed through serial otoendoscopies. Results At the end of the 16-week follow-up, cholesteatoma was present in 13 of 38 (34.2%) ears in the intervention group, and in 7 of 34 (20.6%) in the control group (p = 0.197). When we considered cholesteatoma and its potential precursor, pars flaccida retraction pocket, in a combined way, we verified it in 23 of 38 (60.8%) in the intervention group and in 11 of 34 (32.3%) in the control group (p = 0.016). Conclusions Over the 16 weeks of follow-up, serial otoendoscopies enabled us to evaluate the formation and development of pars flaccida retraction pockets and cholesteatomas in Mongolian gerbils and proved to be an excellent diagnostic tool.