This comparative study disclosed the diverse catalytic activities of Brevibacillus laterosporus on two different azo dyes. It decolorized 100% of Remazol red and 95% of Rubine GFL within 30 and 48 h respectively, under static condition at 50 mg l(-1) dye concentration. Significant increase was observed in azo reductase, NADH-DCIP reductase, veratryl alcohol oxidase and tyrosinase in cells obtained after decolorization of Remazol red; whereas these values were much different with complete inhibition of azo reductase during decolorization of Rubine GFL. The plausible pathway of dye degradation obtained from Gas chromatography-Mass spectroscopy (GC-MS) data confirmed the different metabolic fate of these structurally unidentical dyes. FTIR and HPTLC analysis of extracted metabolites confirmed the biodegradation, while phytotoxicity study assured the detoxification of both the dyes studied. The results obtained in this study suggests, i) sulpho and hydroxyl group present at ortho position to azo group stimulated reduction of azo bond by azo reductase in Remazol red, ii) the same reduction was totally hampered due to presence of ethyl-amino propanenitrile group at para position to azo group in Rubine GFL. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).