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Abstract Microorganisms, especially microscopic filamentous fungi, represent one of the most significant factors influencing the degradation of cultural heritage cellulosic objects. Early detection, identification and characterisation of microbial communities can help select a suitable conservation intervention and, in addition, allow possible control of its effectiveness. A combination of spectral methods and statistical data processing appears to be a suitable alternative to conventional methods for monitoring microbial contamination. The main goal of the presented research was the investigation of the spectral properties of vital and devitalised filamentous fungi Alternaria alternata, Aspergillus niger, Cladosporium herbarum, Penicillium chrysogenum, and Trichoderma atroviride, inoculated on a paper substrate Whatman in two phases—dormant (conidia) and active (mycelium). The combination of the UV-Vis-NIR and NIR Fibre Optics Reflection Spectroscopy (FORS) with the Principal Component Analysis (PCA) was set to determine whether the spectra of vital and devitalised forms of studied samples differ. The obtained results prove differences between the spectra of vital and devitalised forms of filamentous fungi, at least in one studied spectral region. Therefore, UV-Vis-NIR and NIR FORS combined with PCA seems to be a promising tool for monitoring paper-based objects' microbial contamination.