The impact of door opening on cave carbon dioxide (CO2) levels was studied in the Entrance Chamber and the Gallery Chamber of the Balcarka Cave (Moravian Karst, Czech Republic). The effect of door opening differed with cave ventilation modes. Under upward airflow mode, the cave door opening led to the increase of output advective CO2 fluxes from the cave and to the decrease of CO2 levels. This effect was evident especially in the Entrance Chamber near the cave entrance and then suppressed in the Gallery Chamber situated deeper in the cave. Under the downward airflow mode, the cave door opening changed airflow paths and main CO2 sources/fluxes. This resulted in the increase of CO2 level in the Entrance Chamber while the levels in the Gallery Chamber decrease. Modeling indicates that the increase could be result of input advective CO2 fluxes from epikarst (up to 5.9 × 10-2 mol s-1). To reduce the impact on cave microclimate, a careful control of the visiting regime without overlapping of individual doors’ openings is recommended.