Adherence to a biological surface allows bacteria to colonize and persist within the host and represents an essential first step in the pathogenesis of most bacterial diseases. Consequently, the physicochemical properties of the outer membrane in bacteria play a key role for attachment to surfaces and therefore for biofilm formation. Bordetella pertussis is a Gram-negative bacterium that colonizes the respiratory tract of humans, producing whooping cough or pertussis, a highly infectious disease. B. pertussis uses various adhesins exposed on its surface to promote cell-surface and cell-cell interactions. The most dominant adhesin function is displayed by filamentous hemagglutinin (FHA). B. pertussis Tohama I wild-type (Vir+) strain and two defective mutants, an avirulent (Vir-) and a FHA-deficient (FHA-) B. pertussis strains were studied by AFM under physiological conditions to evaluate how the presence or absence of adhesins affects the mechanical properties of the B. pertussis cell surface. Quantitative information on the nanomechanical properties of the bacterial envelope was obtained by AFM force-volume analysis. These studies suggested that the presence of virulence factors is correlated with an increase in the average membrane rigidity, which is largely influenced by the presence of FHA. Moreover, for this system we built a nanoscale stiffness map that reveals an inhomogeneous spatial distribution of Young modulus as well as the presence of rigid nanodomains on the cell surface.