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Carbendazim (CBZ) is a fungicide widely used on different crops, including soybeans, cereals, cotton, tobacco, peanuts, and sugar beet. Excessive use of this xenobiotic causes environmental deterioration and affects human health. Microbial metabolism is one of the most efficient ways of carbendazim elimination. In this work, Rhodococcus qingshengii RC1 and Rhodococcus erythropolis RC9 were isolated from a bacterial community growing in a biofilm reactor acclimated with microbiota from carbendazim-contaminated soil. Sequencing analysis of genomes of both strains revealed the presence of cbmA, the gene coding for the enzyme that hydrolyses carbendazim to produce 2-aminobenzimidazole (2-AB). The alternative gene for the first catabolic step (mheI) was detected by PCR in strain RC9 but not in RC1. Metabolomic analysis by HPLC and LC-MS showed that both strains have the ability to metabolize carbendazim. R. qingshengii RC1 converts carbendazim to 2-AB, the first degradation intermediary, while R. erythropolis RC9 metabolizes the fungicide to its mineralization, probably because R. qingshengii RC1 lacks the hdx gene coding for 2-AB hydroxylase. HRESIMS-MS/MS results indicate that R. erythropolis RC9 metabolizes carbendazim by cleavage of the benzene ring and subsequent formation of 5-formyl-2-hydroxy-4,5-dihydro-1H-imidazole-4-carboxylic acid (X2 C5H6N2O4). The presence of carbendazim metabolites in culture supernatants of strains RC9 and RC1 suggests that both strains contribute to the efficient degradation of carbendazim in nature.