Although the chemical composition of mycobacterial cell walls is well known, the 3D organization of the various constituents is not fully understood. In particular, it is unclear whether the major wall component lipoarabinomannan (LAM) is exposed on the outermost surface or hindered by other constituents such as mycolic acids. To address this pertinent question, we used atomic force microscopy (AFM) with tips bearing anti-LAM antibodies to detect single LAM molecules on Mycobacterium bovis BCG cells. First, we showed the ability of anti-LAM tips to detect isolated, purified LAM molecules. We then mapped the distribution of LAM on mycobacteria, prior to and after treatment with the drug isoniazid. We found that LAM was not exposed on the surface of native cells, pointing to the presence of a homogeneous layer of mycolic acids, whereas it was greatly exposed on isoniazid-treated cells, in agreement with the action mode of the drug. This single-molecule study provides novel insight into the architecture of mycobacterial cell walls and offers promising perspectives for understanding the action modes of antimycobacterial drugs.