The adaptability of soil microbial communities to prolonged periods of drought is influenced by their ability to produce extracellular polymeric substances (EPS) with sufficient water retention properties. Microbial EPS have been extensively investigated as water reservoirs during drought, but it remains unknown how carbon substrate accessibility to soil microbial communities will affect the chemical properties of the EPS they generate, and whether this in turn will alter their water retention characteristics. In this work, we observed that the accessibility of carbon substrates influenced microbial community structure and, consequently, the chemical properties of EPS produced by the microbial communities. Our results demonstrated that an insoluble carbon substrate (i.e., chitin), stimulated microbial communities to produce EPS with measurably better water retention properties in emulated soil microenvironments in comparison to a soluble carbon substrate (i.e., N-acetylglucosamine; NAG). In all, this study demonstrates the importance of carbon substrate accessibility by soil microorganisms in regulating the community structure and consequently, the EPS carbon chemistry, which in turn can greatly influence the adaptability of soil microbial communities to drought. •Carbon substrate accessibility regulates microbial community membership.•The properties of microbial community-produced EPS can be influenced by membership.•Soil microbial community membership influence EPS water retention properties.•Specific carbon substrates may affect microbial community EPS production and properties.