Mycobacterium abscessus is an emerging multidrug-resistant bacterial pathogen causing severe lung infections in cystic fibrosis patients. A remarkable trait of this mycobacterial species is its ability to form morphologically smooth (S) and rough (R) colonies. The S-to-R transition is caused by the loss of glycopeptidolipids (GPLs) in the outer layer of the cell envelope and correlates with an increase in cording and virulence. Despite the physiological and medical importance of this morphological transition, whether it involves changes in cell surface properties remains unknown. Herein, we combine recently developed quantitative imaging (QI) atomic force microscopy (AFM) with hydrophobic tips to quantitatively map the surface structure and hydrophobicity of M. abscessus at high spatiotemporal resolution, and to assess how these properties are modulated by the S-to-R transition and by treatment with an inhibitor of the mycolic acid transporter MmpL3. We discover that loss of GPLs leads to major modifications in surface hydrophobicity, without any apparent change in cell surface ultrastructure. While R bacilli are homogeneously hydrophobic, S bacilli feature unusual variations of nanoscale hydrophobic properties. These previously undescribed cell surface nanodomains are likely to play critical roles in bacterial adhesion, aggregation, phenotypic heterogeneity and transmission, and in turn in virulence and pathogenicity. Our study also suggests that MmpL3 inhibitors show promise in nanomedicine as chemotherapeutic agents to interfere with the highly hydrophobic nature of the mycobacterial cell wall. The advantages of QI-AFM with hydrophobic tips are the ability to map chemical and structural properties simultaneously and at high resolution, applicable to a wide range of biosystems.