AbstractThe objective of this study is to examine microbial behavior by fabricating and applying parallel concentric wrinkled patterns to polydimethylsiloxane (PDMS) substrates. The patterns are generated and subsequently relaxed in a rigid silica layer, resulting in the formation of discernible wrinkles that can be analyzed using electron microscopy and light diffraction. The alignment of E. coli and silica microparticles is facilitated by these structures along the creases. Within these creases, E. coli exhibits enhanced attachment and rotational mobility in regions characterized by hydrophobic cracks. Furthermore, the bacteria demonstrate an increase in velocity when propelled by the PDMS creases, suggesting that these structures are capable of guiding microbial motion. In addition, distinct alignment and growth patterns are observed for E. coli proliferation within the PDMS microchannels under confined conditions. The analysis of bacterial morphology and gene expression in response to chemical stimuli, such as isopropyl‐β‐D‐thiogalactopyranoside (IPTG) gradients, demonstrates that the chemical environment has a substantial impact; filamentous cells aligned along the creases. These results illustrate the efficacy of parallel concentric wrinkled PDMS patterns in establishing regulated conditions for investigating microbial behavior and represent significant contributions to the fields of microbiology and biomedicine.