The intensive use of diclofenac (DCF), a nonsteroidal anti-inflammatory drug, has led to its wide occurrence in the environment. The present study investigated the potential use of the fungal Scytalidium thermophilum laccase for DCF removal and detoxification. Firstly, the culture conditions for the maximum fungal laccase production were optimized with a hybrid design and the obtained conditions were: glucose, 13gL(-1); ammonium tartrate, 7.5gL(-1); yeast extract, 11.5gL(-1); CuSO4, 300 mu M; temperature, 42 degrees C and aeration, 30%. A maximum laccase activity of 8220UL(-1) was detected at 5th day, which was fivefold higher than the result with the initial medium. Secondly and for the first time, the laccase successfully removed 98% of DCF at optimum conditions (37 degrees C, pH 4.5 and DCF 200mgL(-1)) within 8h and without mediators. Genotoxicity, cytotoxicity and phytotoxcity revealed a non toxic metabolites after biologic treatment via S. thermophilum laccase. In fact, hemolytic effect of treated DCF was significantly less than that of untreated DCF. This result was confirmed by morphological variations in erythrocyte shape in both treated and untreated DCF, in comparison to untreated control of red blood cells. In addition, the algal toxicity suggested that treated DCF was significantly less toxic than untreated DCF in chlorophyll contents showing an increase of carotenoids and lycopenes, in comparison to the control culture of D. salina. The effectiveness of S. thermophilum laccase in the DCF biotransformation and its toxicity removal can be considered an interesting option for further environmental applications. [GRAPHICS] .