Infrared spectra of Pseudomonas aeruginosa PAO1 biofilms grown on 40° ZnSe and 40° AMTIR (amorphous material transmitting infrared radiation, Ge33As12Se55) internal reflection elements (IRE) at 26 °C in a flow cell were collected. The depth of penetration of the evanescent wave was sufficient to identify four biofilm stages during the experiment: (1) cell/substratum attachment, (2) lag phase, (3) later-stage growth, and (4) restructuring (e.g. rearrangement, detachment or “erosion”). Our experimental results provide the first detection via a non-perturbative technique of an excess of protein in the neighbourhood of the surface. Our mathematical analysis applied to the data supports the conclusion that our observations cannot be completely accounted for by the changing characteristics of bacterial proteins but, rather, shows that an accumulation of excess protein near the attachment surface is taking place. On the assumption that biofilm cells are approximately of the same size as those of the planktonic culture, this suggests the presence of extracellular proteins. Our data do not support the presence of extracellular nucleic acids near the attachment surface in these biofilms. This conclusion is based on our observation that, during the initial 20 h of development (stage 2 and the beginning of stage 3), biofilms had a higher proportion of protein to nucleic acid than in the latter part of stage 3 and in stage 4. In the restructuring phase this ratio became equal to the ratio determined for planktonic cultures. The decrease of biomass during stage 4 is inconsistent with the physical removal or sloughing-off of cellular aggregates from biofilms within a few micrometres of the substrate surface but, instead, supports a model of gradual cell migration away from it. During this cell migration there is also a gradual loss of extracellular protein. Alternatively, therefore, the restructuring may involve cell lysis and the observed loss of biomass may be due to soluble macromolecules being removed from the neighbourhood of the surface.