Introduction. The course of both the bacterial species and load and the incidence of infection during negative pressure wound therapy (NPWT) are unclear, with published studies presenting contradicting results. Objective. The aim of the study is to assess the changes in both bacterial species and load, as well as the incidence of infection, before and after NPWT in a patient population with a variety of wounds. Methods. Surgical patients 18 years of age or older who needed NPWT were included in this multicenter, prospective cohort study. A wound swab culture was taken before NPWT and either immediately following NPWT or 6 weeks of follow-up. The change of bacterial species, bacterial load, and rate of infection were determined before and after the start of NPWT. Results. In total, 104 patients were analyzed. The number of positive cultures increased from pre- to post-NPWT. The most cultured pathogenic bacterium was Staphylococcus aureus. The bacterial load was moderately higher at the end of NPWT than at the start (P < .0001). It was noted that 2 swabs contained multidrug-resistant bacteria, 1 pre-NPWT and 1 post-NPWT. Prior to NPWT, 26 patients had a wound infection, 5 of which had a persisting infection at the end of the study. Post-NPWT, 14 patients developed a wound infection. Conclusions. The number of S aureus strains and overall bacterial load increased during NPWT, and the incidence of infection remained the same. Further studies should be conducted to determine whether the increase in bacterial load influences other wound outcome parameters.