Core Ideas Extracellular polymeric substances (EPS) sorb to hematite nanoparticle surfaces. High EPS loadings on hematite cause an increase in colloidal stability. Hematite is immobilized in glass bead porous media during transport. Colloidally stable EPS–hematite associates are mobile in glass bead porous media. Colloidal stability and the mobility of mineral nanoparticles largely depend on the properties of the nanoparticle surface. Thus, any modification of the surface due to adsorption will alter the fate of nanoparticles in the environment. In this study, we explored the effect of extracellular polymeric substances (EPS) on the colloidal stability and the mobility of hematite nanoparticles (HNP). Hematite nanoparticles were amended with increasing concentrations of EPS (20, 200, and 500 mg L−1 C) that resulted in EPS‐C to HNP‐Fe mass ratios of 1:5, 2:1, and 5:1 (HNP 1:5, HNP 2:1, and HNP 5:1). With increasing EPS loadings, the isoelectric point of the EPS–HNP associates shifted from 9.8 (pure HNP without EPS) to ?2 (HNP 5:1), resulting in an aggregation of HNP 1:5 with an isoelectric point near the solution pH of 7. In NaCl solution, both the aggregation kinetics and the critical coagulation concentration (CCC) increased with increasing loadings of EPS. Conversely, in CaCl2 solution, the CCC decreased with increasing EPS loadings due to the formation of Ca bridges. The mobility of pure HNP and EPS–HNP associates was explored with transport experiments in closed‐flow mode at neutral pH in a porous medium made of glass beads. Pure HNP were colloidally stable but completely retained in the porous medium due to strong electrostatic interactions between the HNP and the glass surfaces. In contrast, for colloidally stable EPS–HNP associates (HNP 2:1 and HNP 5:1), a persistent mobile fraction and surface attachment was observed. This highlights that EPS association promotes mobility of HNP that were otherwise rendered immobile.