This work aims to study the by-products generated by the anaerobic degradation process of synthetic wastewater containing indigo dye. These by-products were analysed and identified by both high performance liquid chromatography (HPLC) and nuclear magnetic resonance (H-1 NMR). HPLC results showed the dependence of the by-products to the operating conditions. The obtained HPLC chromatogram at the end of the experiments (Run 1) reveals different fractions, with at least eight distinguishable by-products. Increasing the hydraulic retention time from 1 to 5days (Run 5) which corresponding to the start up of the bioreactor caused a significant change of the obtained HPLC chromatogram, with the decrease of the number of these by-products to only 3 ones. H-1 NMR analysis was realised with three representative fractions. The results showed that hypothetical structure of the by-products corresponding to aromatic cycles 1,2-disubstituted and possessing an axial symmetry similar to the phthalate groups