Background: Dental caries commonly occurs during orthodontic treatment because fixed appliances can impede effective oral hygiene practices. This study investigated the effects of fixed orthodontic treatment on dental biofilm maturity and virulence gene (gtfB, ldh, brpA, spaP, luxS, and gbpB) expression. Methods: Dental biofilms and virulence gene expression were determined in 24 orthodontic patients before and after treatment of ≥6 months. A three-tone disclosing gel was used to stain dental biofilm and assess its maturity by its color change—pink (new dental biofilm), purple (mature dental biofilm), and light blue (cariogenic dental biofilm). Gene expression levels were determined using real-time PCR. Results: After fixed orthodontic appliance insertion, the percentage of new dental biofilm decreased, whereas that of cariogenic dental biofilm significantly increased (p < 0.05). There was no significant difference in the percentage of mature dental biofilm (p > 0.05). Fixed orthodontic appliances increased gtfB, ldh, brpA, and gbpB gene expression above 1.5-fold in dental biofilm. In contrast, there was no change in spaP or luxS gene expression after treatment. Conclusions: Fixed orthodontic appliance insertion induced ecological changes and cariogenic virulence gene expression in dental biofilm.