Acellular human dermal allografts have been shown to be effective for soft-tissue implantation. We compared treatment outcomes of tympanoplasty using tragal perichondrium and acellular human dermal allograft (MegaDerm®). In a prospective randomised controlled study, 60 patients scheduled to undergo tympanoplasty were randomly assigned to the autologous tragal perichondrium group (n = 33) or acellular human dermal allograft group (n = 27). Postoperative hearing gain, graft success rate at 1 and 6 months and operation times were compared between groups. Graft success rate, defined as the complete closure of tympanic membrane perforation, did not show any significant intergroup difference (75.8% vs 85.2%, p = 0.519). Air conduction thresholds and air-bone gaps showed significant improvements in both groups; from 38.7 ± 15.9 dB to 30.2 ± 15.6 dB (p < 0.001) and from 17.8 ± 7.3 dB to 11.5 ± 7.0 (p = 0.001) in the autologous tragal perichondrium group, and from 30.4 ± 12.2 dB to 24.5 ± 13.0 dB (p = 0.006) and from 14.3 ± 5.1 dB to 7.6 ± 4.6 dB (p < 0.001) in the acellular human dermal allograft group. The amount of hearing gain (p = 0.31) and closure of air-bone gap (p = 0.863) were not meaningfully different between groups. The mean operation time was significantly lower in the acellular human dermal allograft group (35.2 min vs 27.4 min, p = 0.039). In this prospective randomised controlled study, acellular human dermal allograft was shown to be an effective alternative to tragal perichondrium, with similar graft success rates and postoperative hearing results, but with reduced operation times.