Several reports have concluded that enzymatic debridement based on Bromelain (NX) is selective and efficient. Although clinical trials showed that viable tissue is not damaged at the macroscopic level, the effect on the cellular level is largely unknown. The current study is meant to close this gap by evaluating whether NX has an effect on vital cells of the human dermis on a cellular level. In an experimental in vitro study design, the effect of NX on human keratinocytes, fibroblasts, and macrophages was analyzed. Enzymatic treatment was performed for 4 hours by using either cell culture medium or phosphate-buffered saline as diluting agent for NX. Cell viability and relative cell number in relation to untreated control cells were determined using a resazurin-based assay. In addition, the development of enzyme activity during clinical treatment was analyzed: wound fluid collected from a burn wound at different points of debridement was applied on collagen-elastin disks to prove enzymatic digestion activity. Both keratinocytes and fibroblasts were damaged by NX even at low concentrations. Both cell types showed improved survival when a medium was used for dissolving NX. Macrophages appeared to resist NX treatment more efficiently than the other cell types. In the clinical trial, NX activity in the wound fluid decreased clearly following 4 hours of enzymatic debridement. NX induces toxicity of vital skin cells in vitro. However, macrophages appear to be more resistant against NX treatment in vitro. The inflammatory responses of vital cells in the burn wound itself are likely to inhibit NX activity. The effect of this inflammatory process on NX activity will have to be investigated in future studies.