Introduction: This study was designed to test the hypothesis that thermoplastic retainers influence oral microbial flora during the retention period because they prevent the flushing effect of saliva on dental and mucous tissues. Methods: Twenty-four orthodontic patients finished the study. After debonding, the patients were given thermoplastic retainers (Essix ACE 0.040-in plastic, Dentsply International, York, Pa) for both jaws and instructed to wear them all day. Plaque samples from tooth surfaces and saliva samples were collected from each patient just after debonding (T0), and on day 15 (T1), day 30 (T2), and day 60 (T3) of retention. The jaws were divided into 6 regions, and the data for each region were evaluated separately. Total viable Lactobacillus and Streptococcus mutans colonies were counted, and the numbers of the viable microorganisms were calculated. Results: The numbers of Lactobacillus colonies at T3 were higher than at T0, T1, and T2, and the difference between T0 and T3 was statistically significant (P <0.05). The numbers of S mutans colonies at T3 were higher than at T0, T1, and T2, and the differences between T0 and T1, and T1and T2 were statistically significant (P <0.05). Conclusions: Retention with thermoplastic retainers might create oral conditions conducive to S mutans and Lactobacillus colonization on dental surfaces. Copyright © 2012 by the American Association of Orthodontists.