Aims: To isolate micro-organisms capable of utilizing polyhexamethylene biguanide (PHMB) as a sole source of nitrogen, and to demonstrate biodegradation of the biocide. Methods and Results: Two consortia of bacteria were successfully enriched at the expense of PHMB, using sand from PHMB-treated swimming pools as inoculum. Both consortia were shown to contain bacteria belonging to the genera Sphingomonas, Azospirillum and Mesorhizobium. It was shown that the presence of both Sphingomonas and Azospirillum spp. was required for extensive growth of the consortia. In addition, the Sphingomonads were the only isolates capable of growth in axenic cultures dosed with PHMB. Using a stable isotope (N-15)-labelled PHMB, metabolism of the biocide by both consortia was demonstrated. By comparing the level of N-15 atom incorporation into bacterial DNA after growth on either N-15-PHMB or N-15-labelled NH4Cl, it was possible to estimate the percentage of PHMB biodegradation. Conclusions: The microbial metabolism of nitrogen from the biguanide moiety of PHMB has been demonstrated. It was revealed that Sphingomonas and Azospirillum spp. are the principal organisms responsible for growth at the expense of PHMB. Significance and Impact of the Study: This is the first study to demonstrate the microbial metabolism of PHMB.