The formation of biofilms by diverse bacteria isolated from contaminated soil and groundwater on model substrata with different surface properties was assessed in a multifactorial screen. Diverse attachment phenotypes were observed as measured by crystal violet dye retention and confocal laser scanning microscopy (CLSM). Bulk measurements of cell hydrophobicity had little predictive ability in determining whether biofilms would develop on hydrophobic or hydrophilic substrata. Therefore selected pairs of bacteria from the genera Rhodococcus, Pseudomonas and Sphingomonas that exhibited different attachment phenotypes were examined in more detail using CLSM and the lipophilic dye, Nile Red. The association of Rhodococcus sp. cell membranes with lipids was shown to influence the attachment properties of these cells, but this approach was not informative for Pseudomonas and Sphingomonas sp. Confocal Raman Microspectroscopy of Rhodococcus biofilms confirmed the importance of lipids in their formation and indicated that in Pseudomonas and Sphingomonas biofilms, nucleic acids and proteins, respectively, were important in identifying the differences in attachment phenotypes of the selected strains. Treatment of biofilms with DNase I confirmed a determining role for nucleic acids as predicted for Pseudomonas. This work demonstrates that the attachment phenotypes of microbes from environmental samples to different substrata varies markedly, a diverse range of macromolecules may be involved and that these differ significantly between genera. A combination of CLSM and Raman spectroscopy distinguished between phenotypes and could be used to identify the key macromolecules involved in cell attachment to surfaces for the specific cases studied.