Di (2-ethyl hexyl) phthalate (DEHP) is a plasticizer present in various products, mainly those made with PVC. This phthalate has attracted attention due to its important participation in the contamination of the environment. It has been reported as an endocrine-disrupting compound in mammals. Fusarium culmorum is a phytopathogenic fungus able to degrade DEHP, because it produces esterases, which are enzymes capable to break down ester bonds present in the structure of phthalates. In this research, growth, protein content and esterases activity by biochemical tests and polyacrylamide gel electrophoresis were characterized for F. culmorum grown in DEHP-supplemented (100 and 1500 mg/L) media as the only carbon source in liquid fermentation. F. culmorum showed higher biomass production and esterase activity in medium supplemented with 1500 mg of DEHP/L. Zymography revealed that bands with esterase activity were observed after 24 h and 48 h in media supplemented with 1500 and 100 mg of DEHP/L, respectively. It was shown that DEHP is an inducer of esterases and that this compound was used as carbon and energy sources by F. culmorum. This fungus can secrete specific esterase to breakdown high concentrations of DEHP, being a promising organism for bioremediation of DEHP-polluted environments in both aquatic and terrestrial ecosystems.