Recent investigations on the interactions of bacteria with micro/nanostructures have revealed a wide range of prokaryotic responses that were previously unknown. Despite these advances, however, it remains unclear how collective bacterial behavior on a surface would be influenced by the presence of anisotropic nanostructures with subcellular dimensions. To clarify this, the attachment, aggregation, and alignment of Pseudomonas aeruginosa on orderly subcellular nanogratings with systematically varied geometries were investigated. Compared with a flat surface, attachment and aggregation of bacteria on the nanogratings were reduced by up to 83 and 84% respectively, whereas alignment increased by a maximum of 850%. Using a semiempirical quantitative model, these results were shown to be caused by a lowering of physicochemical attraction between the substrate and bacteria, possible disruption to cell communication, and physical isolation of bacteria that were entrenched in the nanogratings by capillary action. Furthermore, the bacterial attachment level was generally found to be exponentially related to the contact area between the substrate and bacterial cells, except when there were significant deficits in the available contact area, which prompted the bacterial cells to employ their appendages to maintain a minimum attachment rate. Because the contact area for adhesion is strongly dependent on the geometry of the surface features and orientation of the bacterial cells, these results indicate that the conventional practice of using roughness parameters to draw quantitative relationships between surface topographies and bacterial attachment could suffer from inaccuracies due to the lack of shape and orientation information provided by these parameters. On the basis of these insights, design principles for generating maximal and minimal bacterial attachment on a surface were also proposed and verified with results reported in the literature.