Nicosulfuron is a selective herbicide belonging to the sulfonylurea family, commonly used on maize culture. A bacterial strain SG-1 was isolated from an agricultural soil previously treated with nicosulfuron. This strain was identified as Pseudomonas fluorescens and is able to quantitatively dissipate 77.5% of nicosulfuron (1 mM) at 28 °C in the presence of glucose within the first day of incubation. Four metabolites were identified among which ASDM (2-(aminosulfonyl)-N,N-dimethyl-3-pyridinecarboxamide) and ADMP (2-amino-4,6-dimethoxypyrimidine) in substantial proportions, corresponding to the hydrolytic sulfonylurea cleavage. Two-phase dissipation kinetics of nicosulfuron by SG-1 were observed at the highest concentrations tested (0.5 and 1 mM) due to biosorption. The extend and rate of formulated nicosulfuron transformation were considerably reduced compared to those with the pure active ingredient (appearance of a lag phase, 30% dissipation after 10 days of incubation instead of 100% with the pure herbicide) but the same metabolites were observed. The toxicity of metabolites (standardized Microtox® test) showed a 20-fold higher toxicity of ADMP than nicosulfuron. P. fluorescens strain SG-1 was also able to biotransform two other sulfonylureas (metsulfuron-methyl and tribenuron-methyl) with various novel pathways. These results provide new tools for a comprehensive picture of the sulfonylurea environmental fate and toxicity of nicosulfuron in the environment.