Interest in the topics of energy efficiency and thermal comfort integrated with building preservation in historic buildings is growing among researchers. Research has determined that an adequate indoor microclimate should be maintained, avoiding abrupt variations between indoor and outdoor environments which could damage artwork, the building itself, and diminish occupant thermal comfort. The novelty of this research is intended to be the use and validation of a simulation model to determine which natural ventilation scenario delivers the most adequate indoor environmental conditions for occupants, artwork, and building preservation on a case study with very particular aspects such as being a listed historic building in a hot and humid climate during the cooling season. The severity of the local climate during the summer months combined with the heritage preservation requirements, create a research gap that this study attempts to reduce. This article presents a three-dimensional Computational Fluid Dynamics and energy model to evaluate the suitability of natural ventilation in a UNESCO World Heritage Site in San Antonio, Texas. The model was calibrated with real monitored data to obtain reliable results from the indoor environmental conditions, simulating three natural ventilation scenarios and eighteen computational models. The results reveal that the mechanical system operation can be decreased, particularly in spring, when the proposed night ventilation scenario is able to keep air temperatures and relative humidity levels within an optimal range for occupant comfort and building and artwork preservation. The results achieved have led to the development of recommendations that will be valuable for the preventive preservation of such historic buildings, without compromising human comfort. This study reveals that this holistic approach can be implemented using natural ventilation and contributes to reduce mechanical systems operation.