Bacterial biofilms are highly tolerant to antimicrobials and play an important role in the development and spread of antibiotic resistance based on horizontal gene transfer due to close cell-to-cell contact. As an important surface property, topography has been shown to affect bacterial adhesion and biofilm formation. Here, we demonstrate that micrometer-scale surface topographies also affect horizontal gene transfer through conjugation in bacterial biofilms. Specifically, biofilm formation and associated conjugation on poly(dimethylsiloxane) (PDMS) surfaces with 10 μm tall protruding patterns were studied using fluorescently labeled donor and recipient strains of Escherichia coli. The results demonstrate that square-shaped topographic patterns with side length of 20, 50, and 100 μm and interpattern distance equal to or larger than 10 μm promote biofilm formation and conjugation compared to the smooth control. The vertical sides of these topographic features were found to be the "hot spots" for bacterial conjugation compared to the top of patterns and grooves between topographic features. The increase in conjugation frequency on the sides of topographic patterns was attributed to the high cell density of recipient cells at these locations. A motility (motB) mutant of the recipient strain exhibited defects in biofilm formation at the "hot spots" and conjugation, which were recovered by complementing the motB gene on a plasmid. These results also provided guidance for designing surface topographies that can reduce conjugation. Specifically, 10 μm tall hexagon-shaped topographic patterns with side length of 15 μm and interpattern distance of 2 μm were prepared to reduce biofilm formation on the side of protruding patterns and interrupt cell-cell interaction in the grooves. This topography exhibited 85% and 46% reduction of biofilm formation and associated conjugation, respectively, compared to the smooth control.