The human amniotic membrane (hAM) has lately demonstrated interesting potential in its retinal applications. Nowadays, the treatment of persistent macular holes is still a challenge. In this paper, we present a case of a hAM plug used to treat a persistent macular hole, with the purpose of highlighting the critical issues of this new surgical procedure. We performed a 23-G pars plana vitrectomy and inserted a hAM plug (diameter of 1.5 mm) under the neuroretinal margins of the hole (diameter of 657 μm). A complete ophthalmic examination with the assessment of best-corrected visual acuity (logMAR units, ETDRS letter score) and optical coherence tomography (OCT) scans were collected at 1, 4, 6, and 10 weeks after surgery. Visual acuity changed from logMAR 1.0 (1) preoperatively to logMAR 1.0 (2), 1.0 (4), 0.9 (9), and 0.9 (10) at 1, 4, 6, and 10 weeks of follow-up, respectively. The 1 week postoperative OCT did not show neuroretinal adhesion over the plug, but the following visits showed reattachment of the margins with partial neuroretinal ingrowth. These findings suggest that the correct adaptation of the size of the hAM plug to the macular hole is essential to allow the anatomical closure of the hole and potential functional results.