Divalent cations, such as magnesium (Mg2+) and calcium (Ca2+), have been shown to change the biofilm structure and detachment rate. The impact of calcium concentrations on the biofilm morphology, structure, detachment and denitrification efficiency in Denitrifying Fluidized Bed Bioreactors (DFBBRs) was investigated. The DFBBRs were operated on a synthetic municipal wastewater at five different calcium concentrations ranging from the typical Ca2+ concentration of the tap water (20 mg Ca2+/L) to 240 mg Ca2+/L at two different C/N ratios of 5 and 3.5 in phases I and II, respectively for a period of 200 days. The results showed that calcium concentrations played a significant role in biofilm morphology with the detachment rates for R120Ca (bioreactor with a Ca2+ concentration of 120 mg/L), R180Ca, and R240Ca 90% and 70% lower than for R20Ca and R60Ca, respectively. The optimum influent calcium concentration at both organic and nitrogen loading rates was 120 mg Ca2+/L, with higher concentrations exhibiting fractured and weak biofilms. Specific denitrification rates did not change with changing the C/N ratio at elevated Ca2+ concentration bioreactors while with lower Ca2+ concentrations, the specific denitrification rates dropped by 20%-40%. Nutrients and Ca2+ mass balances were closed with reasonable accuracy. Some of the data presented here is published in Eldyasti et al.