Biofilms of different colours that colonize surfaces within karst caves represent a source of nutrients. Yellow microbial biofilms were sampled from three caves in southwestern Slovenia, Dimnice, Krizna jama and Sveta jama. The highest concentration of cultivable microbes and the biggest number of identified bacteria were retrieved from a sample from Sveta jama. Using Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight for bacterial identification showed that different species of Pseudomonas prevailed in all samples. Yellow biofilms showed an absorption peak around 400 nm, and two emission peaks, a major in the blue and a minor in the orange parts of the spectrum when excited at 405 nm. Microbial mats that colonize surfaces are probably frequently overlooked in caves because they are difficult to observe when they have no pigmentation and the contrast with their surroundings is low. Additional studies are needed to aid the understanding of the role of pigmented biofilms and their interactions with underlying substrata in respect of the evolution of substrate micromorphology.