The childcare environment represents an appropriate avenue to support physical activity among preschoolers. The aim of this study was two-fold: (1) to measure the physical activity levels of a sample of preschoolers during childcare hours; and (2) to assess which attributes (e.g., space, equipment, policies) within centre-based childcare environments influenced physical activity. Thirty-one preschoolers from 5 childcare centres across London, Canada participated. Actical accelerometers were worn by participants for one day during childcare hours to assess activity levels using a 15 second epoch length. The Environment and Policy Assessment and Observation instrument was used to conduct a full-day evaluation of the childcare environment. On average, participants engaged in 1.54 min/h of moderate-to-vigorous physical activity and 17.42 min/h of total physical activity. Sedentary opportunities, portable and fixed play equipment, and staff behaviour accounted for 49.3% of the variability in moderate-to-vigorous physical activity and 14.1% of the variability in total physical activity, with sedentary opportunities, fixed play equipment, and staff behaviours displaying an inverse relationship. Results emphasize the critical role the childcare environment plays in supporting physical activity among preschoolers.