SummaryCyanides are highly toxic and produced by various microorganisms as defence strategy or to increase their competitiveness. As degradation is the most efficient way of detoxification, some microbes developed the capability to use cyanides as carbon and nitrogen source. However, it is not clear if this potential also helps to lower cyanide concentrations in roadside soils where deicing salt application leads to significant inputs of ferrocyanide. The question remains if biodegradation in soils can occur without previous photolysis. By conducting a microcosm experiment using soils with/without pre‐exposition to road salts spiked with 13C‐labelled ferrocyanide, we were able to confirm biodegradation and in parallel to identify bacteria using ferrocyanide as C source via DNA stable isotope probing (DNA‐SIP), TRFLP fingerprinting and pyrosequencing. Bacteria assimilating 13C were highly similar in the pre‐exposed soils, belonging mostly to Actinomycetales (Kineosporia, Mycobacterium, Micromonosporaceae). In the soil without pre‐exposition, bacteria belonging to Acidobacteria (Gp3, Gp4, Gp6), Gemmatimonadetes (Gemmatimonas) and Gammaproteobacteria (Thermomonas, Xanthomonadaceae) used ferrocyanide as C source but not the present Actinomycetales. This indicated that (i) various bacteria are able to assimilate ferrocyanide‐derived C and (ii) long‐term exposition to ferrocyanide applied with deicing salts leads to Actinomycetales outcompeting other microorganisms for the use of ferrocyanide as C source.