Hydrogen peroxide (H2O2) solution 1% to 6% has been used for cleaning of wounds for many years but is not recommended nowadays because of its concern about potential tissue damaging effects. The product studied here is a H2O2 cream that has been on the market for many years for treatment and prevention of minor skin infections. The H2O2 is stabilized through embedding in a lipid crystalline structure resulting in a depot cream. The lipids used boost the antimicrobial effect of the hydrogen peroxide and the sustained release system results in a product with long lasting antimicrobial effect in situ. The cream is an excellent alternative to topical antibiotics without the risk for bacterial resistance which is an increasing concern. An increased clinical effect has been observed for wound healing when using this cream and this effect is more than an antimicrobial cleaning effect. The exact role and therapeutic levels of H2O2 is not completely understood even if recent knowhow of H2O2 as part of the signalling messenger in wound healing is available. The studies presented here are aiming to correlate the concentration of endogeneous H2O2 at the wound site and the significance of exogenous hydrogen peroxide in wound healing. H2O2 is in the body a naturally occurring mediator that is released in the inflammatory processes. It could positively affect wound healing by increasing angiogenesis, facilitate fibroblast proliferation and activity, and increase wound constriction. At the same time, excessive H2O2 may be toxic and prevent wound healing. Release of H2O2 from various formulations including 1% solution and cream, 0.5% solution and cream, and 0.25% cream through flow cells over a period of 4 hours using dialysis membranes was determined. The release of H2O2 was proportional to the concentration in the donor formulations. The concentration released to the wound surface was correlated to bridging cell proliferation studies using cultures of L929 mouse fibroblast and MTS-assay for 24 and 48 hours, respectively. The concentrations of H2O2 ranged from 10−3 to 10−10 M, where 10−3 M and 10−5 M H2O2 had a profound antiproliferative effect on the L929 cells, compared to the control without addition of H2O2. Whether H2O2 at the concentrations 10−6 to 10−10 M are beneficial for cell proliferation or not, is currently investigated. In conclusion, there is a steady release rate of H2O2 over the dialysis membrane, and H2O2 can inhibit L929 cell proliferation at high concentrations.