Bivalent ions such as Ca2+ and Mg2+ are known to affect the structural and mechanical properties of biofilms. In order to reveal the impact of Fe2+ ions within the cultivation medium on biofilm development, structure and stability, Bacillus subtilis biofilms were cultivated in mini-fluidic flow cells. Two different Fe2+ inflow concentrations (0.25 and 2.5 mg/L, respectively) and wall shear stress levels (0.05 and 0.27 Pa, respectively) were tested. Biofilm structure was determined daily in situ and non-invasively by means of optical coherence tomography. A set of ten structural parameters was used to quantify biofilm structure, its development and change. Moreover, for each experiment ten replicates were cultivated and analyzed allowing for valid conclusions. Fe2+ addition influenced biofilm development (e.g., biofilm accumulation) and structure markedly. Experiments revealed the accumulation of FeO(OH) within the biofilm matrix and a positive correlation of Fe2+ inflow concentration and biofilm accumulation. Even at elevated shear stress levels this correlation was valid. In more detail, independent of the wall shear stress applied during cultivation over ten days biofilms grew approximately four times thicker at 2.5 mg Fe2+/L compared to low Fe2+ inflow concentrations of 0.25 mg/L. This finding hints on a higher stability of Bacillus subtilis biofilms against detachment when growing at elevated Fe2+ concentrations.