The current study utilized Box-Behnken design of the response surface methodology, aimed at identifying the best levels of particular variables. An experimental design was performed on Chlorella sp. considering initial nitrogen, phosphorus and iron concentration as independent variables. Lipid, biomass, chlorophyll, carbohydrate and protein contents were analysed as response variables. Various physio-biochemical attributes were studied under varied concentration of nutrients in BG11 media, nitrate (NaNO 3 , 10-750 mg mL −1), phosphate (K 2 HPO 4 , 40-120 mg mL −1), iron (ferric ammonium citrate, 3-9 mg mL −1) with an objective to establish the actual potential of Chlorella pyrenoidosa under phototrophic nutrient stress conditions were obtained lipid percentage (35.4 dcw%) and biomass yield (1.89 g L −1). A Prob > F value of < 0.05 and > 0.05 indicated model terms which were significant and non-significant, respectively. The fit of model yielded R 2 values up to 96.25% for lipids and 94.12% for biomass; similar values were obtained for proteins, carbohydrate and chlorophyll. FAME profile of Chlorella pyrenoidosa contained palmitic (C16:0, stearic acid), (C18:0) oleic (C18:1), linoleic C18:2 and linolenic (C18:3) which showed that Chlorella pyrenoidosa possessed a favourable fatty acids profile that can be successfully utilized for biodiesel production.