Childhood obesity remains a problem in the United States. Preschool teachers can help to attenuate it but need to have the confidence or self-efficacy to provide healthy practices. Therefore, the purpose of this study was to (a) develop and validate a preschool teacher self-efficacy tool to examine teachers' confidence in addressing the nutrition and physical activity needs of children in their care and (b) examine relationships between teachers' self-efficacy and knowledge of healthy nutrition and physical activity practices. Instrument design included examination of related literature and review by 12 subject matter experts. Two hundred seventy-three early childhood professionals submitted a 58-item questionnaire with 48 self-efficacy items in four domains of healthy practices for the preschool classroom; the remaining 10 items reflected knowledge of healthy practices. Two hundred fourteen professionals completed the questionnaire. A principle components factor analysis supported a five-factor self-efficacy scale (three of the original domains and one domain split into two factors) with 46 items. Knowledge of healthy practices and age of the professionals predicted overall self-efficacy. Results indicated evidence of validity for this new assessment tool to assess early childhood professionals' self-efficacy toward healthy practices in preschool classrooms.