Fatty acids rich-oil was extracted from freeze-dried fungal biomass of Nigrospora sp. using ultrasound with ethanol as a cosolvent + supercritical CO2. The parameters of temperature (40-80 degrees C), pressure (15-25 MPa), ultrasound pre-treatment time (15-60 min), and the addition of ethanol (30-70 g/100 g biomass) were evaluated on the oil yield and composition. The best oil yield (3.94 wt.%) and total fatty acids (45.5 g/100 g oil) were obtained at 80 degrees C and 25 MPa with 50 wt.% ethanol after pre-treating the biomass for 60 min using 132 W of ultrasound power at 40 kHz. The main fatty acids were oleic, palmitic, linoleic, elaidic and palmitoleic acids. The combination of CO2 under supercritical conditions and ultrasound with ethanol as a pre-treatment of the biomass from Nigrospora sp. concentrated up to 4.0 times fatty acids in the oil when compared to the Soxhlet technique, especially the unsaturated ones.