Nuclear magnetic resonance (NMR)-based metabolomic approach is a high-throughput fingerprinting technique that allows a rapid snapshot of metabolites without any prior knowledge of the organism. To demonstrate the applicability of NMR-based metabolomics in the field of microalgal-based bioremediation, novel freshwater microalga Scenedesmus sp. IITRIND2 that showed hypertolerance to As(III, V) was chosen for evaluating the metabolic perturbations during arsenic stress in both its oxidation states As(III) and As(V). Using NMR spectroscopy, we were able to identify and quantify an array of ∼45 metabolites, including amino acids, sugars, organic acids, phosphagens, osmolytes, nucleotides, etc. The NMR metabolomic experiments were complemented with various biophysical techniques to establish that the microalga tolerated the arsenic stress using a complex interplay of metabolites. The two different arsenic states distinctly influenced the microalgal cellular mechanisms due to their altered physicochemical properties. Eighteen differentially identified metabolites related to bioremediation of arsenic were then correlated to the major metabolic pathways to delineate the variable stress responses of microalga in the presence of As(III, V).