Biofilms are three-dimensional structures formed as a result of microorganism's adhesion on a biotic or abiotic surface. Once a biofilm is established, it is onerous to eradicate it or kill the pathogens therein. Thus, targeting the microbial adhesion process, the initial stage of biofilm formation, is a reasonable approach to avoid challenges associated with subsequently formed biofilms. While many properties of interacting material that play significant roles in initial bacterial adhesion have been widely studied, the effect of surface stiffness on bacterial adhesion was relatively underexplored. In this study, we aimed to investigate the effect of surface stiffness on the adhesion of microbial species found in the oral cavity by employing representative oral bacteria, Streptococcus mutans and Streptococcus oralis, and the fungus, Candida albicans. We compared the adhesion behavior of these species alone or in combination toward various surface stiffness (0.06 – 3.01 MPa) by assessing the adhered and planktonic cell numbers at an early (4 h) adhesion stage under various carbon sources and the presence of conditioning film. Our data revealed that in general, a higher amount of microbial cells adhered to softer PDMS surfaces than stiffer ones, which indicates that surface stiffness plays a role in the adhesion of tested species (either single or co-cultured). This pattern was more obvious under sucrose conditions than glucose + fructose conditions. Interestingly, in monospecies, saliva coating did not alter the effect of surface stiffness on S. mutans adhesion while it diminished S. oralis and C. albicans adhesion. However, in the multispecies model, saliva coating rendered the percentage of all adhered microbes to varied PDMS not distinct. The data provide new insights into the role of surface stiffness on microbial mechanosensing and their initial adhesion behavior which may set a scientific foundation for future anti-adhesion strategies. © 2022 Elsevier B.V.