Aims: Development of the resazurin microplate method (RMM) as a novel test system for the evaluation of the antimicrobial activity of antiseptics and disinfectants. The validated RMM was subsequently applied for the evaluation of hydrogen peroxide (H2O2) and stabilized H2O2 combination products. Methods and Results: The European Committee for Standardization prescribes the plate count challenge test (PCCT) for antiseptic and disinfectant efficacy testing. This protocol was adapted to a microplate-based assay, using resazurin as viability indicator. The RMM was as accurate as the PCCT, had an identical detection limit and showed high intermediate precision. Using the validated RMM, it was shown that H2O2 combined with silver possessed a higher bactericidal and fungicidal activity compared to native H2O2 with and without glycerol. Conclusions: Validation showed that the RMM may replace the PCCT. When applying the RMM, H2O2 combined with silver was clearly a more potent disinfectant compared to H2O2 in killing bacteria and fungi. Significance and Impact of the Study: The RMM is easier to use for antimicrobial efficacy testing of antiseptics and disinfectants. As the RMM is in accordance with the norms of the European Committee for Standardization, it may become a more cost-effective alternative to the more laborious PCCT reference method. H2O2 with silver may replace native H2O2 to increase overall disinfection efficiency.