Ensuring the comfort and health of occupants is the main objective of properly functioning building systems. Regardless of the season and building types, it is the priority of the designers and building managers. The indoor air parameters affect both the well-being and health of users. Furthermore, it could impact the effectiveness of their work and concentration abilities. In hotel facilities, the guests’ comfort is related directly to positive opinions or customer complaints, which is related to financial benefits or losses. The main goal of this study is the analysis of the indoor environmental quality in guests’ rooms, based on the example of a hotel in Poland. The article assesses the variability of air parameters, including temperature, humidity, and carbon dioxide concentrations, and the acceptability of indoor conditions. The research was carried out in November 2020. Based on the collected data, the dynamics of changes of selected air parameters were analyzed. The article analyzes the comfort indicators inside guest rooms, including the Predicted Mean Vote (PMV) and Predicted Percentage of the Dissatisfied (PPD) index. The obtained results were compared with the optimal conditions of use to ensure the guests’ comfort. As the analysis showed, the temperature and humidity conditions are maintained at a satisfactory level for most of the time. It was noticed that the CO2 concentrations temporarily exceeded the value of 2000 ppm in two of the analyzed guests’ rooms, which could cause discomfort to hotel guests. In these rooms, the increase in the volume of ventilation airflow should be considered. The measured parameters dynamically varied over time, and there was no repeatability or clear patterns of variation. This is due to the individual preferences and behavior of users. A detailed analysis is extremely difficult due to the possibility of opening windows by users, the irregular presence of hotel guests in the rooms, and the inability to verify the exact number of users in the room during the measurements.