In the Auschwitz-Birkenau State Museum in Oświęcim, Poland (A-BSM) a new research has been started on the use of vaporised hydrogen peroxide (VHP) as a disinfecting agent. The aims of the study were: to identify microorganisms inhabiting historical cardboard using molecular methods (MALDI-TOF MS, 16S rRNA genes sequencing and next-generation sequencing on Illumna platform); to establish the effectiveness of VHP disinfection (300 ppm, 20 min); and to evaluate the impact of VHP and ethylene oxide (EtO) on the physico-chemical properties of the new and historical cardboard (spectrophotometrical colour measurements, SEM, FTIR and XPS), including changes after accelerated ageing. On the surfaces of the historical objects containing cardboard 104–105 CFU/100 cm2 of bacteria and approximately 103 CFU/100 cm2 of fungi were detected. The predominant were: Bacillus licheniformis, Bacillus megaterium, Micrococcus luteus, Staphylococcus epidermidis, Alternaria alternata, Cladosporium cladosporioides and Penicillium chrysogenum. VHP disinfection under the conditions of performed tests resulted in a reduction of 99.61–100.00% for bacteria and 98.82–100.00% for fungi. The surface morphology, cellulose structure, chemical composition and perceptible to the naked eye colour of the new and historical cardboard were not changed after application of VHP in a tested dose and following EtO disinfection.