Bacterial adhesion is described as a multistep process of interactions between microbes and the substrate, beginning with reversible contact, followed by irreversible adhesion. We explore the influence of substrate zeta potential on adhesion of Mycobacterium smegmatis, a nonpathogenic bacterial model for tuberculosis-causing Mycobacterium tuberculosis and a common foulant of reverse osmosis filtration systems. Substrates having a range of zeta potentials were prepared by coating silica with the polycation, poly(diallyldimethyl ammonium chloride) (pDADMAC), by adjusting the pH of alumina, a pH-responsive material, and by coating silica with a hydrophobic self-assembled monolayer coating of octadecyltrichlorosilane. Our observations using these surfaces demonstrated that adhesion of M. smegmatis increased significantly by more than 200% on the silica-pDADMAC system and more than 300% on alumina substrates, as zeta potential became less negative, and that the variation of pH did not affect adhesion on alumina surfaces. Live and heat-killed bacteria were studied to investigate the contribution of biological response to adhesion with respect to zeta potential. While approximately 60% fewer heat-killed M. smegmatis adhered to pDADMAC-coated silica substrates, the trend of significantly increasing adhesion with less negative zeta potential was still observed. These results show the influence of zeta potential on adhesion of M. smegmatis, which is a separate process from that of the biological response. Across the range of substrate surface chemistries, hydrophobicities, and zeta potentials tested, adhesion of M. smegmatis can primarily be controlled by zeta potential. The bacterial zeta potential was not changed by the various experimental conditions and was -28.3 ± 2.4 mV.