Background: Negative pressure wound therapy (NPWT) has established successfully as a treatment option for acceleration of wound healing in a broad range of clinical indications. Systematic research investigating the bacterial kinetics on wounds is lacking and no studies are available comparing the microbiological difference of repeated instillation of polyhexanide (PHMB) against continuous NPWT without instillation of an antiseptic. The aim of this study was to investigate the bacterial bio-burden on wounds and to measure the bacterial kinetic during application of NPWT with and without intermitted instillation of 0.02% PHMB. Methods: A cohort of 18 patients with chronic (n = 16) and acute (n = 2) wounds was treated either with NPWT alone or with additional intermitted instillation of 0.02% PHMB. Wound healing and bacterial load were assessed at every dressing change. All wounds were closed by split-thickness skin grafts or local Reverdin flaps. According to the manufacturer of the investigated NPWT system (V.A.C. therapy unit, Kinetic Concepts Inc. KCI) wounds were filled with a polyurethane sponge (pore size 0.4-0.6 mm) and occluded with a polyurethane foil (KCI Inc., USA). The NPWT-device was connected to the wound and operated at 100 mm Hg sub-atmospheric pressure utilizing an alternating pressure cycle of 5 min of suction followed by 2 min pause. The automated intermittent instillation lasted for 4-20 s, the exposure time was 20 min. After 60 min of NPWT, the instillation was started again. Results: Seven split-thickness skin grafts, 3 Reverdin-flaps and one combined closure (Reverdin + split-thickness skin grafts) exhibited excellent wound healing after successful transplantation 4 weeks after intervention. The quality of wound granulation was excellent in all wounds, regardless of NPWT application with or without instillation of 0.02% PHMB. The bacterial spectrum remained mostly unchanged after treatment and had no influence on the outcome. Conclusion: It was demonstrated that management of infected wounds with NPWT, with or without instillation of 0.02% PHMB, did not reduce the number of bacteria on wounds. Bacteria proliferate below a NPWT dressing and increase in numbers. Independently of bacterial kinetics, all wounds showed excellent granulation and healed with no complications. This observation challenges the concept that bacterial bioburden correlates with impaired wound healing.