There is a growing interest for the mixotrophic cultivation of microalgae using sustainable natural carbon sources. Chitin from shrimp waste was hydrolyzed using chitinase from Trichoderma asperellum and was exploited for the mixotrophic cultivation of microalgae. Chitin saccharification was optimized using Box-Behnken design to produce 50.41% (w/w) of reducing sugars using chitin, 0.83% (w/v), chitinase, 2.92 (v/v), temperature, 40 °C and time, 4.48 h. The biomass productivity of Tetradesmus obliquus and Aphanocapsa sp. was promoted to 39.1 and 40.57 mg L⁻¹ day⁻¹ in the presence of 0.5 g L⁻¹ of N-acetylglucosamine oligomers (GlcNAc), which was estimated to be ∼1.9 and ∼2-folds higher than the autotrophic conditions, respectively. Modified Logistic kinetic model confirmed higher growth rates and shortened lag-time of the mixotrophic cultures. The lipid productivity of both microalgae species was >1.5-times higher than the autotrophic conditions. Nitrate removal from the culture medium was found to be an effective strategy to increase both lipid content and lipid productivity for T. obliquus, but this strategy was not effective for Aphanocapsa sp. Fatty acid analysis revealed an increase of saturated fatty acids under mixotrophic cultivation for both microalgae species. Additionally, several biodiesel evaluation parameters indicated superior characteristics for the mixotrophic culture compared to the autotrophic conditions. Accordingly, mixotrophic growth of microalgae using GlcNAc as a new, sustainable carbon and nitrogen source opens a new avenue for biofuel production with an effective waste conversion into valuable commodities.