The potential impact of cyanobacteria and microalgae on the weathering of calcareous tesserae from a Roman mosaic of the II Century CE has been followed through in vitro experiments. Laboratory tests were carried out by inoculating mosaic tiles with single strains of Cyanobacteria or Chlorophyta to evaluate the roles of pioneer phototrophic microrganism on the resulting architecture of biofilms. The interaction between tesserae and strains was assessed at the whole substratum and micrometer scales, by image analysis and Confocal Laser Scanning (CLS) microscopy, respectively. The biofilm surface coverage on each tessera varied from 19% (Fischerella ambigua) to 97% (Microcoleus autumnalis). Cyanobacteria showed a better growth on calcareous tesserae, whereas the only green alga attaining a superficial coverage higher than 50% was Coelastrella rubescens. CLS microscopy evidenced two different types of spatial arrangement of the phototrophic organisms on the tesserae, that were defined as compact or porous, respectively. In the first one was measured a reduced number of empty spaces between cells or filaments, whereas in the second type, a reticulate texture allowed the presence of numerous empty volumes. The colonization processes observed are an intrinsic characteristic of each strain. We have proposed a colonization index IC as a sensible tool to describe, in a quantitative way, the pioneering attitude of each photosynthetic microorganism to colonize lithic substrates under laboratory conditions.