A protocol for isolation of protoplast from the mycelium of Fusarium pallidoroseum, a potential biocontrol agent of water hyacinth, was standardized. Stable and more medium sized protoplasts were obtained from 18 h old mycelium at 3 h after incubation, using 80 mg of lytic enzyme from Trichoderma harzianum, in 4 ml of 0.01 M citrate buffer (pH 5.85) containing 0.6 M KCl and 5 mM DTT. The protoplasts were regenerated in the regeneration medium.