Autologous bio-engineered dermo-epidermal skin substitutes are a promising treatment for large skin defects such as burns. For their successful clinical application, the graft dressing must protect and support the keratinocyte layer and, in many cases, possess antimicrobial properties. However, silver in many antimicrobial dressings may inhibit keratinocyte growth and differentiation. The purpose of our study was to evaluate the effect of various wound dressings on the healing of a human hydrogel-based dermo-epidermal skin substitute in preparation for the first-in-human clinical trials. Human dermo-epidermal skin substitutes approved for clinical trials were produced under good manufacturing practice conditions, transplanted onto immuno-incompetent rats, and dressed with either Vaseline Gauze™ (Kendall Medtronic, Minneapolis, USA), Suprathel® (PolyMedics Innovations GmbH, Denkendorf, Germany), Urgotul® SSD (Urgo Medical, Shepshed, United Kingdom), Mepilex® AG (Mölnlycke Health Care, Gothenburg, Sweden), or Acticoat™ (Smith&Nephew, Baar, Switzerland). Grafts were assessed clinically for take, epithelialization, and infection at 10 and 21 days post-transplantation, and histologically at 21 days. There were three subjects each in the Vaseline Gauze™ and Suprathel® groups, and four subjects each in the Urgotul® SSD, Mepilex® AG, and Acticoat™ groups. For all samples, the take rate was 100% and the expected keratinocyte number, epithelialization and epidermal stratification were observed. All of the dressings in the current study were well tolerated by our human dermo-epidermal skin substitute. The tolerance of the silver-based dressings is particularly relevant given the high risk of bacterial contamination with large skin defects, and provides pivotal information as we embark on clinical trials for this novel skin substitute.