Background: Little is known about patterns of sedentary behavior (SB) and physical activity among preschoolers. Therefore, in this observational study patterns of SB and moderate-to-vigorous physical activity (MVPA) were examined in detail throughout the week in preschool-aged boys and girls. Methods: A sample of 703 Melbourne preschool children (387 boys; 4.6 plus or minus 0.7 y) were included in data analysis. SB and MVPA data were collected using accelerometry over an eight-day period. Percentage of time per hour in SB and in MVPA between 08:00 h and 20:00 h was calculated. Multi-level logistic regression models were created to examine the hour-by-hour variability in SB and MVPA for boys and girls across weekdays and weekend days. Odds ratios (OR) were calculated to interpret differences in hour-by-hour SB and MVPA levels between boys and girls, and between weekdays and weekend days. Results: The highest SB levels co-occurred with the lowest MVPA levels from the morning till the early afternoon on weekdays, and during the morning and around midday on weekends. Besides, participation in SB was the lowest and participation in MVPA was the highest from the mid afternoon till the evening on weekdays and weekend days. The variability across the hours in SB and, especially, in MVPA was rather small throughout weekdays and weekends. These patterns were found in both boys and girls. During some hours, girls were found to be more likely than boys to demonstrate higher SB levels (OR from 1.08 to 1.16; all p < 0.05) and lower MVPA levels (OR from 0.75 to 0.88; all p < 0.05), but differences were small. During weekends, hour-by-hour SB levels were more likely to be lower (OR from 0.74 to 0.98; all p < 0.05) and hour-by-hour MVPA levels were more likely to be higher (OR from 1.15 to 1.50; all p < 0.05), than during weekdays, in boys and girls. Conclusion: Entire weekdays, especially from the morning till the early afternoon, and entire weekend days are opportunities to reduce SB and to promote MVPA in preschool-aged boys and girls. Particularly weekdays hold the greatest promise for improving SB and MVPA. No particular time of the week was found where one sex should be targeted.