Sterilization of municipal waste for a raw material for the production of refuse-derived fuel and to protect surface and ground waters against biological contamination during transfer and storage creates a lot of problems. This paper evaluates the antimicrobial potential of non-equilibrium plasma in relation to the selected groups of microorganisms found in humid waste. The proposed research is to determine whether mixed municipal waste used for the production of alternative fuels can be sterilized effectively using low-temperature plasma generated in a gliding arc discharge reactor in order to prevent water contamination and health risk for working staff. This work assesses whether plasma treatment of raw materials in several process variants effectively eliminates or reduces the number of selected groups of microorganisms living in mixed municipal waste. The presence of vegetative bacteria and endospores, mold fungi, actinobacteria Escherichia coli, and facultative pathogens, i.e., Staphylococcus spp., Salmonella spp., Shigella spp., Enterococcus faecalis and Clostridium perfringens in the tested material was microbiologically analyzed. It was found that the plasma treatment differently contributes to the elimination of various kinds of microorganisms in the analyzed raw materials. The effectiveness of sterilization depended mainly on the time of raw materials contact with low-temperature plasma. The results are very promising and require further research to optimize the proposed hygienization process.