Children spend the majority of their weekdays in classrooms that often have low indoor air quality and limited financial resources for the initial and running costs of mechanical ventilation systems. Designing effective natural ventilation (NV) systems in schools is difficult due to the intense use of the classroom spaces and the dependence of NV on building geometry and outdoor conditions. Building thermal and airflow simulation tools are fundamental to predict NV system performance in the design phase. These predictions of these tools must be validated (preferably with data from real buildings). This paper presents a set of detailed measurements of buoyancy driven natural DV systems of three classrooms located in two buildings in Lisbon (Portugal). The rooms are located in two educational buildings, a kindergarten and a university, and have different buoyancy driven natural DV systems (with and without chimneys). The experimental measurements are used to validate a three-node DV model implemented on the open-source thermal building simulation software EnergyPlus. The validation results show that the building thermal simulation model tested is able to predict bulk airflow rate with an average error of 16%. In addition, a good agreement is also obtained for the vertical temperature prediction: an average error of 4% corresponding (average deviation of 0.7 °C). Analysis of the kindergarten rooms results revealed, that as expected, increasing chimney height from 1 to 4 m has a significant positive impact in NV system performance. The performance of natural DV systems depends on the number of thermal plumes in the room. For the same sensible heat load, increasing the number of plumes lowers the average occupied zone air temperature and increases the bulk airflow rate. In light of the complexity of the cases tested, NV with uncontrolled boundary conditions, the results of the comparisons performed between measurements and simulations should contribute to increase confidence in the use of EnergyPlus to simulate buoyancy driven natural DV systems.