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The microbial spoilage and correlated surface changes of a cellulose nitrate negative film, a gelatine–silver positive print, a cardboard frame and a cellulosic envelope dated back to 1938–1940 was assessed by means of molecular methods and scanning electron microscopy (SEM). Materials characterisation was obtained with Raman, Infrared and Electronic dispersion spectroscopies. DNA was extracted from bacteria and fungi, amplified through PCR oriented to bacterial 16S rRNA gene and fungal internal transcribed spacer (ITS), and clone libraries were constructed for each investigated material. The ITS fungal cloning was able to detect a bigger spectrum of species respect to bacterial one. Correspondence between molecular results and SEM observations was used to address the cause of biodeterioration to single species, and to map the presence of different organisms in separate niches. This investigation highlighted a co-occurrence of both bacteria and fungi on most of the substrata, and a spatial partitioning according to the different photographic materials. Moreover, for the first time, the effects of a biological attack on glassine paper were documented. [ABSTRACT FROM AUTHOR]