Bivalent cations are known to affect the structural and mechanical properties of biofilms. In order to reveal the impact of Fe ions within the cultivation medium on biofilm development, structure and stability, biofilms were cultivated in mini-fluidic flow cells. Two different Fe inflow concentrations (0.25 and 2.5 mg/L, respectively) and wall shear stress levels (0.05 and 0.27 Pa, respectively) were tested. Mesoscopic 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. The study focused on characterizing biofilm structure and development at the mesoscale (mm-range). Therefore, biofilm replicates ( = 10) were cultivated and analyzed. Three hypotheses were defined in order to estimate the effect of Fe inflow concentration and/or wall shear stress on biofilm development and structure, respectively. It was not the intention to investigate and describe the underlying mechanisms of iron incorporation as this would require a different set of tools applied at microscopic levels as well as the use of, i.e., omic approaches. Fe 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 Fe inflow concentration and biofilm accumulation. In more detail, independent of the wall shear stress applied during cultivation, biofilms grew approximately four times thicker at 2.5 mg Fe/L (44.8 µmol/L; high inflow concentration) compared to the low Fe inflow concentration of 0.25 mg Fe/L (4.48 µmol/L). This finding was statistically verified (Scheirer-Ray-Hare test, ANOVA) and hints at a higher stability of biofilms (e.g., elevated cohesive and adhesive strength) when grown at elevated Fe inflow concentrations.