In this research, standard growth medium of Nannochloropsis oculata was manipulated with 5-, 10-, 15- and 20-fold increased (15.8, 31.6, 47 and 63 mg/L) and decreased (0.63, 0.316, 0.21, and 0.15 mg/L) chelated FeCl3. The standard medium (control group) contained 3.16 mg/L FeCl3. The results revealed that the highest iron concentration (63 mg/L) partially restricted photosynthetic efficiency (0.67 µg/L). Also, increasing in Fe concentration from 3.16 (control) to 63 mg/L led to significant increase in the total carotenoid contents. The highest lipid accumulation was measured in the cultured algae at 0.15 mg/L FeCl3 (p < 0.05). The highest C16:0 and C18:2 fatty acids values were obtained at 0.316 and 0.15 mg/L Fe concentrations, respectively. C18:1 and C18:3 fatty acid had the maximum value in the media at 0.21 and 0.15 mg/L Fe-depletion (p < 0.05). Moreover, the C20:5 (EPA) had the highest value (24.21%) at 0.63 mg/L FeCl3. The highest and lowest triacylglycerol values were obtained in 0.316 and 63 mg/L FeCl3. These results suggest that Fe limitation in N. oculata growth media with 10- and 20-fold decrease (0.316 and 0.15 mg/L) was the most effective in improving lipid and fatty acid accumulation.