Background: No in vitro surgical study has evaluated the time-dependent contamination of surgical suction tips compared with controls. Our purpose was to determine the difference in suction tip bacterial contamination rates between suction-positive and suction-negative tips. Materials and Methods: A matched-pair analysis of the contamination of surgical suction tips over a six-hour period was performed in two clean operating rooms. One suction tip was connected to standard wall suction (suction-positive group), with a matched control tip not connected to wall suction (suction-negative group). At time zero and then at hourly intervals for six hours, the distal 3 cm of suction tips were removed, placed in nutrient broth for 48 hours, then plate cultured. One hundred tips were collected for each time interval. Results: Eighty-two of 700 (11.7%) suction tips had bacterial contamination. Sixty-three (18.0%) of 350 suction-positive tips were contaminated, with 19 (5.4%) of the 350 suction-negative tips contaminated (χ 2 = 26.7, p < 0.001). Suction tip contamination was time-dependent with the first significant difference between groups occurring after two hours of continuous suction (χ 2 = 4.0, p = 0.04). Contamination rate in the suction-positive group increased significantly after one hour compared with time-zero controls (χ 2 = 7.1, p = 0.008). There was no significant difference in frequency of positive cultures over time in the suction-negative group compared with time-zero controls. Conclusions: This is the first controlled laboratory study suggesting a time-dependent increase in positive suction tip cultures. From our data, operating room staff should have an awareness that suction tips represent a potential source of bacterial concentration. We recommend that when not in use, suction tip valves be closed if this feature is available, that hosing be manipulated to cease suction when not needed, that suckers be disconnected from tubing, or that suckers be exchanged at frequent intervals. Doing so may reduce bacterial contamination on the suction tip.