The theory of fresco as the standard painting technique in Roman wall paintings, formulated in the mid-1960s by Paolo Mora and Laura Mora and Paul Philippot, has enjoyed general acceptance among specialists to the present day. However, the fresco theory is based on a series of postulates—such as the feasibility of fresco on pontate, the presence of giornate, or the use of alkali-sensitive pigments—which appear to be unsupported by physical or experimental evidence and which would require further assessment in order to validate the theory. Additionally, chemical analyses of Roman murals have identified the presence of various types of organic binders. A comparative study of these analyses reveals a remarkable difference between results obtained by the different researchers. The clustering of results by research group appears highly unlikely, suggesting the possible presence of confounding factors which could lead to distorted results. These factors might be related both to the difficulty in extracting ancient paint media and the influence of biological contamination. If the extraction method used is not adequate for an ancient binding medium, then part of the binder may not be extracted, producing an incomplete characterization of its composition. The lack of data on both the efficiency of the methods used and the possible influence of microbial contamination in the organic material may cause uncertainty in the degree of reliability of the different results obtained, fomenting uncertainty around the techniques used in ancient paintings as well as the treatments suitable for their conservation. Therefore, in the analysis of ancient paint media, the implementation of a protocol of assessment of both the efficiency of the extraction methods and the influence of biological contamination seems advisable.Graphical abstract.