Diclofenac (2-[(2,6-Dichlorophenyl)amino]benzeneacetic acid) is a non-steroidal anti-inflammatory drug. Due to excessive use of diclofenac, this drug has been detected in surface water, ground water and drinking water. In our study, four fungal strain Trametes trogii, Aspergillus niger, Yarrowia lipolytica and Phanerochaete chrysosporium were investigated in terms of diclofenac degradation potential. Trametes trogii was found to be the most efficient strain with 100% diclofenac degradation rate. Two hydroxylated diclofenac metabolites have been identified in culture medium. Crude laccase from T. trogii almost completely removed diclofenac with 97% removal in 48 h. We suggest that the degradation of diclofenac depends on the cytochrome P450 enzyme system and laccase activity. After 24 h incubation decrease in toxicity of diclofenac was confirmed by Microtox test.