Iron-oxidizing bacteria (FeOB) can successively populate low-nutrient aquatic environments and adapt to a broad concentration range of alkaline earth metals, with optimum conditions widely differing from one species to another. For the most abundant known FeOB genera Gallionella and Leptothrix, there is a lack of reports on substrate affinity for calcium and magnesium and necromass assimilability. Single nutrient and combined affinity for Ca and Mg of a wild Gallionella ferruginea isolate and a Leptothrix cholodnii strain as well as growth of heterotrophic L. cholodnii on necromass of autotrophic G. ferruginea were determined by cell density measurements. G. ferruginea responds with Monod-shaped preferences and thus favors waters rich in Ca and Mg. Maximum growth occurred at Ca concentrations five times above those of commonly used modified mineral Wolfe’s medium. L. cholodnii showed a Monod-shaped preference in the low concentration range and an inhibitory response to increasing hardness: concentrations >2 mM Ca or >0.6 mM Mg allow only 50 or 75%, respectively, of maximum specific cell densities. Considering the concentration range with a Monod-shaped response (for L. cholodnii only lower concentration range), both FeOB show a type I independent colimitation for Ca and Mg with lower requirements of Mg than Ca. On a C-limited medium containing G. ferruginea necromass as the only C-source, L. cholodnii cell counts were higher by two orders of magnitude compared to pyruvate medium. Thus, the necromass may serve as a primary C-source for heterotrophic FeOB and other heterotrophic bacteria with technical and economical relevance.