Aim To determine whether caries risk factors, including cariogenic bacterial levels and salivary function, can be used to identify orthodontic patients who develop white spot lesions (WSL). Methods This prospective case-control study comprised 50 patients 11-17 years of age, including 25 controls and 25 cases who developed new WSL during treatment. WSL, oral hygiene and fluorosis were evaluated from intraoral photographs. The biofilm was assessed with bacterial cultures and adenosine triphosphate (ATP) bioluminescence. Salivary analyses were performed to determine the pH of saliva and flow rates. A survey was used to assess snacking frequency, oral hygiene and fluoride utilization. Results There were no between-group pretreatment differences in WSL. Cases reported eating sugary foods significantly more often than the controls. There was a significant decline in oral hygiene during treatment, with no significant between-group difference. There also were no statistically significant between-group differences in the amount of saliva, buffer, ATP bioluminescence and bacterial levels. Both groups showed lower than normal buffer capacity and high bacterial levels. Conclusion Cases had greater sugar intake between meals than controls. ATP bioluminescence, Streptococcus mutans levels with Saliva Check Mutans, and salivary factors do not identify patients who develop WSL.