Bacterial surface-associated proteins play essential roles in mediating pathogen–host interactions and represent privileged targets for anti-adhesion therapy. We used atomic force microscopy (AFM) to investigate, in vivo, the binding strength and surface distribution of fibronectin attachment proteins (FAPs) in Mycobacterium bovis bacillus Calmette-Guérin (BCG). We measured the specific binding forces of FAPs (∼50 pN) and found that they increased with the loading rate, as observed earlier for other receptor–ligand systems. We also mapped the distribution of FAPs, revealing that the proteins are widely exposed on the mycobacterial surface. To demonstrate that the proteins are surface-associated, we showed that treatment of the cells with pullulanase, an enzyme possessing carbohydrate-degrading activities, or with protease, an enzyme that conducts proteolysis, led to a substantial reduction of the FAP surface density. A similar trend was also noted following treatment with ethambutol, an antibiotic which inhibits the synthesis of cell wallpolysaccharides. The nanoscale analyses presented here complement traditional proteomic and molecular biology approaches for the functional analysis of surface-associated proteins, and may help in the search for novel anti-adhesive drugs.