Purpose: To describe the intraoperative characteristics of epiretinal human amniotic membrane grafts used during macular hole (MH) repair under various tamponade conditions, as assessed using intraoperative optical coherence tomography (OCT). Methods: This consecutive case series included all patients who underwent human amniotic membrane implantation for persistent or recurrent MH at the Cleveland Clinic Cole Eye Institute between 2024 and 2025. Intraoperative OCT images were reviewed for the presence of subgraft fluid at up to 3 time points: under balanced salt solution or perfluorocarbon liquid (PFO), after initial fluid–air exchange, and after meticulous fluid–air exchange with targeted aspiration. Results: A total of 4 eyes were included (MH size range: 280–920 µm). Intraoperative OCT showed greater subgraft fluid under balanced salt solution compared with PFO, under which only trace fluid was observed. After initial fluid–air exchange, residual subgraft fluid persisted. Although meticulous aspiration further reduced the fluid, the amount remained greater than that observed under PFO. Conclusions: This study characterizes intraoperative OCT findings of epiretinal human amniotic membrane grafts under various tamponade agents to guide surgeons in optimizing intraoperative graft positioning and minimizing graft slippage during this complex procedure.