Atomic force microscopy (AFM) was used in tapping mode to probe morphological changes in surface-confined cells resulting from adhesion-modifying chemicals. Bacteria (Burkholderia cepacia G4 and Pseudomonas stutzeri KC) were exposed to Tween 20, heparin, disodium tetraborate, sodium pyrophosphate, low ionic strength water, lysozyme/ethylenediaminetetraacetic acid (EDTA), and 3-(4-morpholino)propanesulfonic acid sodium salt (MOPS) buffer las a control), and the surface topography of the cells was examined after exposure to each chemical. Cells were attached to glass slides for AFM imaging by a new method of cross-linking carboxyl groups on the bacterial surfaces with amine groups that had been coupled to glass slides. Topographic images, phase images, traces of surface topography, and analyses of surface roughness were performed on all samples. We are not aware of any studies besides the present one in which phase imaging has been used on bacteria. Height traces illustrated the effect of different chemical treatments on the cells by showing whether the topography of the cell was altered by the different treatments. The surface roughness was quantified in terms of the root-mean-square (RMS) average of the height deviations. All of the treatment chemicals except disodium tetraborate caused higher RMS values to be measured for G4 and KC. Disodium tetraborate flattened the cells and, therefore, resulted in slightly lower or equal RMS values as the control (MOPS buffer). RMS values were correlated with the qualitative shapes of the cells. Lysozyme/EDTA, sodium pyrophosphate, and disodium tetraborate produced the most damage to cellular morphology, as observed by topographic images and surface traces, and decreased cellular viability. These data show that AFM operated in tapping mode can provide a useful method for investigating the consequences of bacterial exposure to surface-modifying chemicals.