Tracheoesophageal voice prostheses need to be replaced due to increased airflow resistance or retrograde leakage of fluid into the trachea as a consequence of biofilm formation. Previous in vitro studies show a change of aerodynamic features of biofilm covered voice prostheses after removal of the prostheses out of the patient. To assess these changes in an in situ situation, aerodynamic characteristics were measured within 45 patients at the beginning and at the end of the wearing process of the Provox 2 voice prosthesis. As a consequence, the influence of biofilm formation on aerodynamic characteristics can be evaluated. In the majority of cases, leakage through the prosthesis was the reason for replacement. No differences were found in the total flow, volume range and intratracheal pressure (ITP) of the voice prostheses measured. The airflow resistance of biofilm covered prostheses was significantly reduced compared to new clean prostheses. However, no correlation was found between the extent of biofilm and the different aerodynamic features measured. Biofilm formation on the Provox 2 is responsible for both reduction in airflow resistance and leakage through the prosthesis by deterioration of the silicone rubber material.