In the current study, the authors investigated the effects of human-induced disruption in a subterranean stable environment containing valuable Palaeolithic paintings and engravings (Ardales Cave, Southern Spain) using a double analysis approach. An environmental monitoring system was installed in the cave to record temperature, relative humidity, carbon dioxide (CO2) and radon (222Rn) concentrations in air. In the same stations, an aerobiological sampling was conducted to quantify the level of airborne microorganisms. The combination of different methods allowed us to detect the extent of human induced changes, confirming that these can be very hazardous in certain cave areas that should be apparently outside the scope of human disturbances, either by their remoteness to the visitor entrance or by being briefly visited. The detection of evident anomalies in the environmental parameters and airborne microorganism concentration in the cave area housing the high density of paintings and engravings helps to control human disturbances and supports the direct application of this double approach for cave management purposes.