This study presents a novel flocculant for harvesting Chlorella vulgaris as model species for freshwater microalgae based on cellulose nanocrystals (CNCs), synthesized from a renewable and biodegradable resource. Cationic pyridinium groups were grafted onto CNCs by two separate one-pot simultaneous esterification and nucleophilic substitution reactions. Both types of modified CNCs were positively charged in the pH range 4-11. Both reactions yielded CNCs with a high degree of substitution (up to 0.38). A maximum flocculation efficiency of 100% was achieved at a dosage of 0.1 g g−1 biomass. In contrast to conventional polymer flocculants, cationic CNCs were relatively insensitive to inhibition of flocculation by algal organic matter. The present results highlight the potential of these new type of nanocellulose-based flocculants for microalgae harvesting.