Door openings in the operating room (OR) have been hypothesized to increase OR environmental contamination. This study measured average colony-forming units (CFU) in the OR as a function of door openings and other potentially important variables. Bacterial settle plates were placed inside and outside of laminar airflow (LAF) by both exit doors, on the instrument table, and on the back instrument table (if applicable) for 48 orthopedic and general surgery procedures. CFU data were paired to Staphylococcus aureus colonization status, door openings, surgery duration, time of day, OR location, number of staff, use of warming devices, temperature, and humidity. The number of door openings in the OR and surgery duration were significantly associated with increased CFU in the OR overall and outside of LAF. However, under LAF conditions, only the number of OR personnel was significantly associated with increased CFU.