No earlier study has investigated the microbiology of negative pressure wound therapy (NPWT) foam using a standardized manner. The purpose of this study is to investigate the bacterial load and microbiological dynamics in NPWT foam removed from chronic wounds (>3 months). To determine the bacterial load, a standardized size of the removed NPWT foam was sonicated. The resulting sonication fluid was cultured, and the colony-forming units (CFU) of each species were enumerated. Sixty-eight foams from 17 patients (mean age 63 years, 71% males) were investigated. In 65 (97%) foams, ≥ 1 and in 37 (54%) ≥2 bacterial types were found. The bacterial load remained high during NPWT treatment, ranging from 104 to 106 CFU/ml. In three patients (27%), additional type of bacteria was found in subsequent foam cultures. The mean bacterial count ± standard deviation was higher in polyvinyl alcohol foam (6.1 ± 0.5 CFU/ml) than in polyurethane (5.5 ± 0.8 CFU/ml) (p = 0.02). The mean of log of sum of CFU/ml in foam from 125 mmHg (5.5 ± 0.8) was lower than in foam from 100 mmHg pressure (5.9 ± 0.5) (p = 0.01). Concluding, bacterial load remains high in NPWT foam, and routine changing does not reduce the load.