Intraoperative tissue or fluid culture remains the gold standard in diagnosing periprosthetic infection. However, an organism is not always isolated from intraoperative cultures. We asked whether preoperative antibiotics interfered with the isolation of organisms from intraoperative tissue samples. We hypothesized organisms isolated from preoperative joint aspirate correspond to those cultures from intraoperative specimens. We retrospectively reviewed 171 patients undergoing TKA, diagnosed with periprosthetic infections from 2000 to 2005, who had a positive preoperative aspiration culture. The details of any antibiotics given to the patients preoperatively were documented. Seventy-two of 171 patients received preoperative antibiotics before surgery. Intraoperative culture was negative in nine of the 72 patients who received antibiotics: a false-negative rate of 12.5%. An organism could not be isolated from intraoperative samples in eight of the 99 patients who did not receive preoperative antibiotics: a false-negative rate of 8%. We observed no difference in the incidence of false-negative cultures between the two groups. The organisms isolated from the aspirate culture were identical to their intraoperative counterparts in 97% of the cases. Administration of preoperative antibiotics to patients with a positive preoperative joint aspirate did not interfere with the isolation of the infecting organism from intraoperative culture samples more than when antibiotics were withheld.