During the pandemic of Covid-19, ventilation rate of buildings and especially in spaces with high occupancy like classrooms, presents high research interest. The ventilation strategies, combined with the use of masks, contribute to the decrease of the infection risk of Covid-19. Also, ventilation improves Indoor Air Quality (IAQ), contributing to the good health of the users and potentially influences their thermal comfort. In the proposed work, the experimental investigation of the ventilation’s adequacy in naturally ventilated classrooms located at the University of Western Macedonia, in Kozani, Greece, took place. Measurements include thermal comfort parameters, as well as IAQ ones, namely carbon dioxide (CO2), volatile organic compounds (VOCs), aldehydes and ozone (O3). The air exchange rates were determined according to the tracer gas decay and equilibrium analysis methods, using CO2 as tracer gas, while simulations analysis using appropriate computational approaches was applied. The results between tracer gas method and simulation analysis were compared, allowing the validation of the adopted models. Given that for both approaches natural ventilation proved to be inadequate, different simulated scenarios of ventilation, including natural and mechanical configuration, were investigated; the relation of ventilation rates to IAQ and thermal comfort was investigated. Moreover, the infection risk, given the determined or simulated IAQ, was assessed, according to relevant approaches.