The article deals with the problem of the effects of using a wooden church on thermal and humidity conditions forming inside. Religious services in the studied site were provided several times a year. The building was not used in the remaining time. The analysis of the effects of the frequency of religious services and the number of people at the services on the formation of temperature and humidity conditions in the wooden church is provided in this paper. The effect of the presence of people in the church on CO2 concentration fluctuations was also studied. Analysis of the results showed that external conditions have the greatest effect on internal microclimate substitutions. The presence of people affects temporary fluctuations in internal parameters. Both the number of people attending the service and the time of year are important. An indoor air temperature amplitude of 9.4 °C was recorded during the winter period. The CO2 level in the church during the service exceeded the limit value of 1000 ppm, reaching 1800 ppm in the extreme case, which could result in decreased comfort for people. The high CO2 concentration may have been caused by a lack of effective ventilation in the building. The obtained simulation results showed a high agreement of the theoretical data with the measurement results (correlation 0.91). The analysis of three simulation variants showed that the people attending the services have a significant share in the gains of thermal energy inside the church. In order to meet the assumptions for variant 1, there are no requirements to start the heating system, assuming a similar schedule of services.