Cocamidopropyl betaine (CAPB) is an amphiphilic surfactant commonly used in a variety of personal care products and in some technical applications. The aim of the study was to obtain bacteria that utilized CAPB from a sample of municipal activated sludge, and to investigate the possible role such bacteria performed in surfactant degradation. The CAPB (300 mg l−1) degradation experiments involved the application of two isolated strains. Whilst tests in a mineral medium containing ammonium salt as a nitrogen source revealed almost complete mineralization of the compound in both strains during 4 days, the same process required more than 29 days of incubation under nitrogen-free conditions. Degradation assays and a series of growth tests with and without the source of nitrogen showed that Pseudomonas sp. FV proved to be the primary degrader of CAPB, capable of utilizing the alkyl chains of the surfactant. The other strain, Rhizobium sp. FM, ensured the degradation of intermediates originating from the primary biodegradation stage and, in the absence of ammonium salt, provided a supply of nitrogen to its microbial partner.