The aim of the study was to optimise operating parameters of a diode laser (810 nm), that could be used to effectively disinfect very small, microbiologically contaminated spots on historical cellulosic objects from the Auschwitz-Birkenau State Museum, and thus limit unnecessary treatment of whole objects. The scope included: assessment of microbiological contamination of the historical materials using culture-dependent (MALDI-TOF MS, 16S rRNA sequencing and macro- and microscopic observations) and culture-independent (NGS sequencing) molecular methods; optimisation of laser irradiation based on the assessment of biocidal effectiveness against 14 microbial strains and determination of changes in colour, and evaluation of the impact of optimal laser variant on the optical (colour measurement spectrophotometry), morphological (SEM), structural (FTIR) and chemical (XPS) properties of the model and historical materials. The small spots were colonised with microorganisms harmful to the objects and posing a threat to human health. The best variant of laser parameters was: 0.3 W of power, continuous wave mode, 2 min of exposure and 2 repetitions, which resulted in 92.17–100.00% reduction in the number of bacteria and complete elimination of 5 out of 6 strains of filamentous fungi, and at the same time it did not have an adverse effect on the colour, morphology, structure and degree of oxidation of the tested materials. A slight increase in the surface polarity was not related to cellulose, but protective substances.