The perception of comfort in seminar rooms significantly affects the performance, concentration, and productivity of its users. This research investigates the impact of the ventilation system, natural ventilation or mechanical ventilation, on user comfort in seminar rooms. Six indoor climate parameters were recorded in identical rooms with natural and mechanical ventilation and were subsequently evaluated from the users’ perspective. A statistical analysis was conducted to compare objective measurement data with subjective perceptions of the indoor climate among the target population. This study aims to determine whether mechanical and natural ventilation systems provide equivalent comfort levels or lead to significant differences in user perceptions. The results from this research provide valuable insights into the impact of ventilation systems on perceived comfort and the mutual interactions between individual factors. Both mechanical and natural ventilation systems created comparable indoor climate conditions in the seminar rooms under consideration. Furthermore, the comfort levels were found to be equivalent for both ventilation systems from the users’ point of view. However, users in rooms with natural ventilation reported higher air velocities to be more pleasant than those in mechanically ventilated spaces. These findings can be used to optimize seminar rooms in terms of perceived comfort, potentially enhancing the performance, concentration and productivity of students.