Aims: To investigate the effect of relative gas humidity on the inactivation efficiency of a cascaded dielectric barrier discharge (CDBD) in air against Aspergillus niger and Bacillus subtilis spores on PET foils. Methods and Resutls: The inactivation kinetics as a function of treatment time were determined using synthetic air with different relative humidity as the process gas. Spores of A. niger and B. subtilis respectively were evenly sprayed on PET foils for use as bioindicators. In the case of A. niger, increased spore mortality was found at a high relative gas humidity of 70% (approx. 2 log(10)). This effect was more evident at prolonged treatment times. In contrast, B. subtilis showed slightly poorer inactivation at high gas humidity. Conclusions: Water molecules in the process gas significantly affect the inactivation efficiency of CDBD in air. Significance and Impact of the Study: Modifying simple process parameters such as the relative gas humidity can be used to optimize plasma treatment to improve inactivation of resistant micro-organisms such as conidiospores of A. niger.