Purpose TNP in the form of V.A.C. dressings have been successfully used to enhance chronic wound healing. An in vitro model of the chronic wound biofilm was created to test the effects of TNP with and without concurrent antimicrobial drug instillation on biofilms.Method A P.aeruginosa biofilm model was constructed in vitro. Continuous TNP and TNP combined with varying frequencies of betadine instillation were tested on the biofilm model for 24 hours. Outcome parameters measured included viable bacterial count, fluorescent microscopy, confocal laser scanning microscopy, and electron microscopy. Statistical analysis was performed using ANOVA and MANOVA in SPSS 13.0.Results Bacterial biofilms, when exposed to TNP alone, showed a 42% decrease in viable count; while exposure to betadine alone showed a reduction of up to 84%. Combined TNP and antiseptic instillation increased the bacteriocidal effect to 99.7% (p < 0.001).Exposure to TNP also resulted in a decrease in biofilm thickness, and diffusion distance, with an increase in surface area‐to‐volume ratio (p = 0.005, 0.002, and 0.009 respectively).Conclusion TNP alone showed a modest effect on the reduction of bacterial biofilm. More importantly it appeared to compress and fragment the overall biofilm architecture. This physical deformity in turn led to more effective drug penetration into the highly resistant biofilm community. The result was an approximate 100 fold enhancement of bacteriocidal effect when betadine instillation was combined with TNP as compared to betadine alone.