Carbon dioxide (CO2) insufflation in the cardiothoracic wound cavity is used in open-heart surgery for prevention of arterial air embolism. The objective of this study was to investigate if CO2 insufflation may influence the rate of airborne contamination of the cardiothoracic wound. This was studied in a cardiothoracic wound cavity model that contained two 9 cm blood agar plates. Contamination rates were compared between a control without insufflation and insufflation with: (1) a thin open-ended tube or a gas-diffuser, (2) air or CO2, and (3) CO2 flows of 5 or 10 L/min. CO2 insufflation at 5 L/min with an open-ended tube resulted in a contamination rate almost four times that of the control (P = 0.01), whereas with the gas-diffuser the contamination rate decreased (P = 0.01). With the gas-diffuser, air insufflation at 5 L/min markedly reduced the contamination rate compared with the control (P < 0.001), but was less protective than CO2 insufflation at the same flow (P < 0.001). With both gases, the contamination rate was particularly low close to the gas-diffuser (P < 0.001). Increasing the CO2 flow from 5 to 10 L/min reduced the average contamination rate in the model from 30% to 22% (P < 0.001) of the control. At a CO2 flow of 10 L/min the contamination rate within 9 cm of the gas-diffuser was 14% of the control. Intraoperative wound ventilation with CO2 using a gas-diffuser may not only prevent air embolism, but may also significantly reduce the risk of airborne contamination and postoperative wound infection in cardiac surgery. In contrast, insufflation with an open-ended tube substantially increases these risks.