The biofilms of Staphylococcus aureus and Escherichia coli have been investigated by atomic force microscopy (AFM). The usage of complex approach of semi-contact AFM mode has shown that the S. aureus and E. coli biofilms represent an organized community of microorganisms. Semicontact error mode and phase imaging mode investigation of extracellular matrix of S. aureus and E. coli biofilms, confirms dimensions of bacteria and biofilms.