The formation of biofilms is a universal bacterial survival strategy. Biofilms occur on inert and living supports in natural environments and in industrial installations. Given their importance in various industrially relevant areas and in human health, numerous investigations have focussed on the particular physiology of these fixed microorganisms. It is now well recognised that bacteria present in biofilms behave quite differently from their planktonic counterparts. In particular, biofilm organisms are far more resistant to antimicrobial agents than are planktonic organisms. The mechanisms involved in the resistance of biofilm bacteria to antimicrobials are complex and still not fully understood. One of the hypotheses that suggested to explain the increased resistance of biofilms to antimicrobial agents assumes the existence of significant differences in gene expression. Although the expression of a limited number of genes appears to be altered during biofilm growth, a number of proteomics studies have revealed large physiological differences between free-living and biofilm bacteria. Moreover, multiple phenotypes were identified during the different stages of biofilm development. This review presents recent data on protein expression in sessile microorganisms that support the existence of a specific metabolic behaviour of biofilm bacteria.