Extracellular polymeric substances (EPS) are ubiquitous in the soil and water environment and interact strongly with mineral surfaces. However, these interactions and their impacts on the behavior and fate of minerals remain poorly understood. Here, for a better understanding of the colloidal stability of minerals in the environment, we investigated the aggregation of goethite (α-FeOOH) nanoparticles (NPs) in the presence of EPS from Bacillus subtilis under different environmental conditions (pH, ionic strength and ionic valence). Results showed that the aggregation processes of goethite NPs are determined by the solution chemistry, and the colloidal stability of goethite NPs is strongly influenced by the addition of EPS. In the absence of ionic strength, the addition of EPS promotes the aggregation of goethite NPs only when the pH (pH = 6) is less than the point of zero charge for the goethite nanoparticles (pH ≈ 8). In the presence of ionic strength, the aggregation rate of goethite NPs increases with increasing concentration of NaCl, NaNO and NaSO solutions, and after the addition of EPS solution, the critical coagulation concentrations (CCC) of goethite NPs are increased from 43.0, 56.7 and 0.39 mM to 168.0, 304.9 and 126.2 mM in the three electrolyte solutions, indicating that the addition of EPS inhibits the aggregation of goethite NPs. While in NaPO solution, when the concentration of NaPO solution ranged from 0 to 1 mM, the aggregation rate of goethite NPs increases first, followed by a decrease, and with the concentration of NaPO solution exceeding 1 mM, the aggregation rate of goethite NPs increases again, due to the charge screening by sodium counter ions. This study provides a fundamental understanding of the behavior of goethite NPs in natural soil and water environments.