Microalgal cultivation systems for energy production are not yet economically viable due to the low yield of lipids and biomass. In this study, we propose a chemical approach, using exogenous bioactive molecules for improving algal growth, lipid content and the production of high-value alpha-linolenic acid (ALA), using Chlorella protothecoides as a model. Based on our findings, among the cytokinin and auxin classes, Kinetin increased the biosynthesis of ALA by 26.5% at 1 ppm concentration, and indole-3-butyric acid at 2 ppm improved the biomass and lipid productivity up to 29.15 ± 2.02 g m− 2 day− 1, and 6.69 ± 0.56 g m− 2 day− 1, respectively. Methyl jasmonate as a stress-related phytohormone also showed a positive stimulatory effect on growth of C. protothecoides, and the biomass productivity was increased up to 25.26 ± 1.48 g m− 2 day− 1 at the concentration of 5 ppm (lipid content: 219.66 ± 13.20 mg g− 1 dw; lipid productivity: 5.61 ± 0.25 g m− 2 day− 1; ALA: 0.81 ± 0.16 g m− 2 day− 1). A difference in the composition of fatty acids, due to the type and concentrations of stress-related phytohormones was observed, and under the influence of stress-related phytohormones, the biosynthesis of saturated fatty acids increased, and polyunsaturated fatty acids decreased in C. protothecoides. Butylated hydroxyanisole, at a concentration of 0.05 ppm, increased the ALA content by 60.5%. Our study shows that the proposed bioactive molecules could increase the biomass productivity of C. protothecoides at a lower cost compared to the use of acetate as a carbon source.