Congo red is one of the best known and used azo dyes which has two azo bonds (-N=N-) chromophore in its molecular structure. Its structural stability makes it highly toxic and resistant to biodegradation. The objective of this study was to assess the congo red biodegradation and detoxification by Aspergillus niger. The effects of pH, initial dye concentration, temperature, and shaking speed on the decolorization rate and enzymes production were studied. The maximum decolorization was correlated with lignin peroxidase and manganese peroxidase production. Above 97% were obtained when 2 g mycelia were incubated at pH 5, in presence of 200 mg/L of dye during 6 days at 28°C and under 120 to 150 rpm shaking speed. The degraded metabolites were characterized by using LC-MS/MS analyses and the biodegradation mechanism was also studied. Congo red bioconversion formed degradation metabolites mainly by peroxidases activities, i.e., the sodium naphthalene sulfonate (m/z = 227) and the cycloheptadienylium (m/z = 91). Phytotoxicity and microtoxicity tests confirmed that degradation metabolites were less toxic than original dye.