Historic photographs are a precious cultural heritage. Despite this, there is a lack of studies on the microorganisms that are responsible for their biodeterioration and the underlying mechanisms. The aims of this study were twofold: (a) assess microorganism diversity on historic photographs using, for the first time, high-throughput sequencing on the Illumina platform; (b) assess the suitability of new methods for mass spectrometric analysis of metabolites on photographs, namely laser ablation-remote-electrospray ionization mass spectrometry imaging (LARESI MSI) in selected reaction monitoring mode (SRM), and surface-assisted laser desorption/ionization mass spectrometry (MS) and mass spectrometry imaging (MSI), on silver nanoparticle enhanced target (109AgNPET MS and MSI). Samples of 19th century Polish silver-gelatin photographs on baryta paper (paper substrate with layer of barium sulphate), obtained by the gelatin developing-out process, were studied. These photographs (after incubation in temperature: 25 ± 2 °C, relative humidity: 80%, time: 60 days) were contaminated with culturable bacteria at concentrations between 3.33 × 101 and 1.60 × 102 CFU/cm2 and fungi at concentrations between 1.57 × 105 and 9.93 × 105 CFU/cm2. Twenty-two phyla of bacteria and one of Archaea were identified on the photographs. Bacteria belonging to Proteobacteria predominated (relative abundance 75.25–84.94%); followed by Firmicutes (1.04–12.61%) and Actinobacteria. Fungi identified belonged mainly to Ascomycota. The dominant bacteria identified included: Mesorhizobium, Burkholderia, Delftia, Enhydrobacter, Paenibacillus, Saccharopolyspora and Olsenella. The dominant fungi belonged to the genera Aspergillus, Alternaria, Penicillium, Chaetomium, Talaromyces, Fusarium. These predominant microorganisms may be responsible for the biodeterioration of the photographs. Direct laser/desorption ionization mass spectrometry could be useful for the reconstruction of heavily damaged photographs. The LARESI MSI method was used to detect metabolites of microbial origin (organic acids and mycotoxins) on the surface of historic photographs, which are compatible with the analysis of fungal biodiversity. The biggest advantage of presented methods is that the tested photographs were not modified in any way before the tests which can be carried out in situ.