Abstract A potent extracellular fibrinolytic enzyme was obtained from cultures of the imperfect fungus Fusarium semitectum under certain growth conditions. Nitrate addition to cultures increased enzyme production. The enzyme showed a versatile proteolytic activity against several protein substrates including casein, gelatin, haemoglobin, bovine serum albumin, and fibrin from both buffalo and human sources. Optimal fibrinolysis occurred at pH values around 7.0. The fibrinolytic activity exhibited marked heat stability in enzyme samples heated to 60 °C, and retained more than 40% of its activity in samples heated to 100 °C for 10 min. Fibrinolysis proceeded optimally in the temperature range between 50–60 °C. Copper ions significantly activated the enzyme. Other biochemical properties are also reported.