Night ventilation methods have been used in educational buildings to guarantee indoor air quality at the beginning of occupied periods. A typical method has been to pre-start ventilation 2 h before the space usage. Another selection has been to ventilate a building continuously during the night with a minimum airflow rate that can dilute material emissions. In this study, the pre-started, continuous, and intermittent ventilation methods were compared by assessing indoor air quality in field measurements. The daytime ventilation was operating normally. The test periods lasted for 2 weeks. Indoor air quality was assessed by measuring the total volatile organic compounds and microbial concentrations using the quantitative polymerase chain reaction method. Additionally, the thermal conditions, carbon dioxide, and pressure differences over the building envelope were measured. The results show that the night ventilation strategy had negligible effects on microbial concentrations. In most cases, the indoor air microbial concentrations were only a few percent of those found outdoors. The averaged concentration of total volatile organic compounds was at the same level with all the night ventilation methods at the beginning of the occupied periods in the mornings. The concentrations reached a minimum level after 2-h ventilation. The concentrations of total volatile organic compounds were higher during the day than at night. This reveals that space usage had the largest effect on the total volatile organic compounds. Generally, the results show that continuous night ventilation does not significantly affect the biological and chemical contaminants. Consequently, a 2-h flushing period is long enough to freshen indoor air before occupancy.