The appearance of crusts or patinas on surfaces of stone monuments are indicative signs of weathering. In many cases, microorganisms are mainly responsible for stone decay, giving rise to the formation of characteristic biodeteriorative patinas, called biofilms. In the present work, “in vitro” biofilm formation on sandstone, limestone, and granite block samples by Penicillium frequentans strains isolated from two Spanish cathedrals is demonstrated. Spore suspensions of P. frequentans strains were inoculated on each block sample of unaltered stone material cited above. Biofilms of 1–2 mm thickness were formed on each of the three rock samples, and analysed by means of Fourier-transform infrared spectroscopy, scanning electron microscopy and X-ray mapping (energy-dispersive X-ray spectrometry, KEVEX system). In the three cases, biofilms were principally composed of: fungal mycelium, mineral particles released from the stony substratum, and newly formed organic salts such as oxalate and citrate. These biofilms enhance and accelerate the deteriorative process of rocks due to the loss of stone material (biopitting and mineral grains captured by mycelium) and to alteration of the mineral crystalline networks (cation release by organic acids).