This paper investigates the microclimate in a conference room with thermal stratification, taking as a case study the chapel of Villa San Saverio, now the seat of the “Scuola Superiore” of the University of Catania (Italy). Surveys of the former chapel were conducted to monitor air temperature and relative humidity. Subsequently, the investigation relied on numerical simulations of a simplified computational fluid dynamics (CFD) model built with the DesignBuilder v7.0 software and validated by comparison with measured values. Simulations were then carried out considering three different scenarios: the current state without any HVAC system and two possible HVAC system configurations providing both air conditioning and ventilation. The results show that, from a comfort perspective, a lightweight radiant floor heating system, assisted by an appropriate ventilation system for air renewal placed at the floor level near the occupants, is preferable to floor-level fan coils and high ventilation channels. Furthermore, this was also confirmed by a preliminary energy analysis of the two HVAC options, where the ventilation effectiveness of the winter period, the temperature of the water the emitters are fed, the consequent COP value of the heat pump, and the electricity consumption were taken into consideration.