Microbes employ a variety of strategies to adhere to abiotic and biotic surfaces, as well as host cells. In addition to their surface physicochemical properties (e.g. charge, hydrophobic balance), microbes produce appendages (e.g. pili, fimbriae, flagella) and express adhesion proteins embedded in the cell wall or cell membrane, with adhesive domains targeting specific ligands or chemical properties. Atomic force microscopy (AFM) is perfectly suited to deciphering the adhesive properties of microbial cells. Notably, AFM imaging has revealed the cell wall topographical organization of live cells at unprecedented resolution, and AFM has a dual capability to probe adhesion at the single-cell and single-molecule levels. AFM is thus a powerful tool for unravelling the molecular mechanisms of microbial adhesion at scales ranging from individual molecular interactions to the behaviours of entire cells. In this review, we cover some of the major breakthroughs facilitated by AFM in deciphering the microbial adhesive arsenal, including the exciting development of anti-adhesive strategies.