To investigate how the thickness and extracellular electron transfer (EET) capabilities of microbial biofilms influence the reduction of ferric iron [Fe(III)]-containing minerals, we utilized four strains of with varying biofilm thicknesses and EET capabilities. These strains were engineered by modulating intracellular levels of dinucleotide second messengers. We systematically investigated the capacity of biofilms formed by four strains to reduce different Fe(III)-containing minerals including ferrihydrite, goethite, and lepidocrocite. By growing the biofilm on the Fe(III) mineral-coated slides, our results showed that the strains forming thin biofilms on surfaces of Fe(III) minerals exhibited faster Fe(III) reduction rates compared to those with thick biofilms. Transcriptomic analyses revealed the upregulation of the genes encoding bacterial EET-involved proteins in the thin biofilms, highlighting the significant role of these proteins in reducing Fe(III)-containing minerals by biofilms. Furthermore, genetic characterization identified the participation of two novel -type cytochromes (-Cyts), GSU1996 and GSU2513, in the reduction of Fe(III)-containing minerals by biofilms. The results from this study provide an improved understanding of mineral-microbe interaction. is a predominant species within biofilm communities that facilitate iron reduction, a process essential for the biogeochemical cycling of iron and other elements. However, the specific properties of biofilms crucial for iron reduction remain unclear. By manipulating intracellular levels of dinucleotide second messengers to generate strains with varying biofilm properties, this research reveals that thinner biofilms exhibit superior rates of ferric iron [Fe(III)] mineral reduction compared to thicker biofilms. This finding highlights the vital role of proteins involved in extracellular electron transfer (EET) in enhancing the reduction of Fe(III)-containing minerals. The study further identifies two novel -type cytochromes, GSU1996 and GSU2513, as important contributors to this process. These discoveries not only advance our understanding of microbial iron reduction but also offer new perspectives on the interactions between biofilms and mineral surfaces, potentially informing future research and applications in biogeochemical cycling and bioenergy.