A novel analytical method using HPLC-MS/MS operating in selected reaction monitoring (SRM) for evaluation of fungi efficacy to decolorize Remazol Brilliant Blue R (RBBR) dye solution was developed, validated and applied. The method shows high sensibility allowing the detection of 4.6 pM of RBBR. Four fungal strains were tested in liquid medium, three strains of Aspergillus (Aspergillus aculeatus, Aspergillus flavus and Aspergillus fumigatus) and Phanerochaete chrysosporium. All fungi were able to degrade the dye, with efficiencies ranging from 40% for P. chrysosporium up to 99% for A. flavus during a 30-day incubation period. During the experiment, increased accumulation of degradation products was observed in A. flavus cultures containing RBBR. Through the use of full scan HPLC-MS technique it was possible to propose the biogenesis of the microbial metabolic degradation pathway. Screening using microorganisms and RBBR may be hereafter used to investigate microbial biodegradation of high toxicity molecules such as dioxins. (C) 2012 Elsevier Ltd. All rights reserved.