In clinical practice, diagnosis of wound infection is based on the classical clinical signs of infection. When infection is suspected, wounds are often swabbed for microbiological culturing. These methods are not accurate (clinical judgment in chronic wounds) or provide results after several days (wound swab). Therefore, there is an urgent need for an easy‐to‐use diagnostic tool for fast detection of wound infection, especially in chronic wounds. This study determined the diagnostic properties of the enzymes myeloperoxidase, human neutrophil elastase (HNE), lysozyme and cathepsin‐G in detecting wound infection when compared to wound swabs. Both chronic and acute wounds of 81 patients were assessed through clinical judgment, enzyme analysis and wound swab. Three promising enzyme models for detecting wound infection were identified. A positive test was defined as: at least one enzyme positive after 30 minutes (model 1), lysozyme and HNE positive after 30 minutes (model 2), myeloperoxidase positive after 5 minutes, and HNE or lysozyme positive after 30 minutes (model 3). All models were significant (p≤0.001). There was no correlation between clinical judgment and wound swab, indicating the need for novel diagnostic systems. Enzyme analysis is fast, easy to use and superior to clinical judgment when compared to wound swabs.