Objective: The bacterial composition and distribution were evaluated in acute standardized epidermal wounds and uninjured skin by a molecular in situ technology benchmarked to conventional culturing. This was done to reveal whether bacterial biofilm is present in acute wounds. Approach: On the buttock of 26 healthy volunteers, 28 suction blisters were made and de-roofed. Four wounds were biopsied immediately after wounding, whereas the remaining 24 wounds were treated daily with sterile deionized water and covered with a moisture-retaining dressing. On day 4 post-wounding, swabs were obtained for culturing from the wounds and adjacent skin, and the wounds including adjacent skin were excised. Tissue sections were stained with peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH) probes, counterstained by 4′,6-diamidino-2-phenylindole, and evaluated by confocal laser scanning microscopy (CLSM). Results: No bacterial aggregates were detected at day 0. At day 4, coagulase-negative staphylococci (CoNS) were the sole bacteria identified by CLSM/PNA-FISH and culturing. CoNS was isolated from 78% of the wound swabs and 48% of the skin swabs. Bacterial aggregates (5-150 μm) were detected by PNA-FISH/CLSM in the split stratum corneum and fibrin deposits at the wound edges and in the stratum corneum and the hair follicles of the adjacent skin. The bacterial aggregates were more common (p = 0.0084) and larger (p = 0.0083) at wound edges than in the adjacent skin. Innovation: Bacterial aggregates can establish in all wound types and may have clinical significance in acute wounds. Conclusion: Bacterial aggregates were observed at the edges of acute epidermal wounds, indicating initiated establishment of a biofilm.