Microalgae have received significant interest as a potential feedstock for the production of biofuels, food, and feed. The effects of temperature and nitrogen limitation on the growth, proximate composition, and fatty acid profile of Nannochloropsis sp. were studied using solvent-oil extraction aided by ultrasonic process as a promoter of microalgae cell disruption. Experiments included a control algal culture grown in F medium (F) at 25°C and four cultures grown in F with half the original concentration of sodium nitrate (F/2) at 25°C, 30°C, 35°C, and 40°C. The results showed that temperature had significant effects on the cell density and growth rate of Nannochloropsis sp. The highest values were obtained when microalgae cultured in F at 25°C, whereas the lowest were obtained in F/2 at 35°C. The protein, carbohydrate, and lipid contents were the highest in F at 25°C and had an increasing tendency in F/2 from 25°C to 30°C; however, the lowest values were obtained in F/2 at 35°C. Both the temperature and nitrogen limitation highly affected the fatty acid profile of Nannochloropsis sp. The proportion of polyunsaturated compounds decreased with increasing temperature from 25°C to 35°C, whereas it increased with nitrogen limitation. These results generate alternatives in Nannochloropsis sp culture conditions that allow have qualitatively best products in relation to their potential use in food and biofuels.