We successfully isolated Rhodococcus sp. D-1, an efficient carbendazim-degrading bacterium that degraded 98.20% carbendazim (200 ppm) within 5 days. Carbendazim was first processed into 2-aminobenzimidazole, converted to 2-hydroxybenzimidazole, and then further mineralized by subsequent processing. After genomic analysis, we hypothesized that D-1 may express a new kind of enzyme capable of hydrolyzing carbendazim. In addition, the effect of the biodegradable biosurfactant rhamnolipid on the rate and extent of carbendazim degradation was assessed in batch analyses. Notably, rhamnolipid affected carbendazim biodegradation in a concentration-dependent manner with maximum biodegradation efficiency at 50 ppm (at the critical micelle concentration, CMC) (97.33% degradation within 2 days), whereas 150 ppm (3 CMC) rhamnolipid inhibited initial degradation (0.01%, 99.26% degradation within 2 and 5 days, respectively). Both carbendazim emulsification and favorable changes in cell surface characteristics likely facilitated its direct uptake and subsequent biodegradation. Moreover, rhamnolipid facilitated carbendazim detoxification. Collectively, these results offer preliminary guidelines for the biological removal of carbendazim from the environment. [ABSTRACT FROM AUTHOR]