Literature data on the effect of calcium on biofilm structures induced a preliminary study. The effect of calcium removal by water softening (<1.0 mg Ca2+.L−1) under real-world drinking water conditions on biofilm formation was studied in a pilot plant with reverse osmosis (RO) membranes and in a laboratory-scale biofilm production unit (BPU) with plasticized polyvinyl chloride (PVC-P) and glass as substratum. The results showed a halving of the exponential biofouling rate in the RO membrane and also a halving of the exponential carbohydrate (CH) production rate in the biofilm on PVC-P and glass in the BPU by softening of the water. In PVC-P biofilms, softening did not affect adenosine tri-phosphate (ATP) production and bacterial species composition (terminal restriction fragment length polymorphism analysis). At low substrate concentrations in glass and RO membrane biofilms softening reduced significantly ATP and CH production and changed the species composition on the membrane. The importance of the two hypothesized physical or physiological mechanisms as causes for the observed Ca2+ effect on biofilm formation and the effect of Ca2+ concentration on those, needs further studies.