The purpose of this prospective clinical trial was to evaluate the success rate of our newly developed surgical technique for fat graft myringoplasty (FGM). We also aimed to assess the correlations between the size of perforations and closure rates, as well as the results of re-gained hearing in successful cases with respect to sizes of perforations. Thirty consecutive patients with persistent tympanic membrane perforation were included in this study. All patients underwent fat graft myringoplasty with a newly developed technique. Treatment success was defined as an intact tympanic membrane (TM) at the 3-month follow-up visit. Bone conduction (BC) and air conduction (AC) thresholds at the frequencies of 0.5, 1, 2, 3, and 4 kHz were recorded preoperatively and at the 3rd month postoperatively. The range of perforation sizes among the 30 patients was 7 mm in 13 of the patients (43 %), 6 mm in 8 of the patients (27 %), and 5 mm in nine of the patients (30 %). Closure of tympanic membrane perforations with this new technique was achieved in 27 out of the 30 patients, with a success rate of 90 %. The average of the air-bone gap (dB) was similar in preoperative and postoperative periods for 5- and 6-mm sized perforation groups (p = 0.09 and p = 0.49). However, in the 7-mm sized perforation group, the average of the air-bone gap (dB) was higher in the preoperative period than the postoperative period (p = 0.004). The average of the air-bone gap (dB) for all patients was higher in the preoperative period than the postoperative period (p = 0.001). In conclusion, FGM performed with this technique may be used as an alternative for the closure of tympanic membrane perforations larger than 5 mm.