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The elastic modulus of single microfibrils from tunicate (Halocynthia papillosa) cellulose was measured by atomic force microscopy (AFM). Microfibrils with cross-sectional dimensions 8 × 20 nm and several micrometers in length were obtained by oxidation of cellulose with 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) as a catalyst and subsequent mechanical disintegration in water and by sulfuric acid hydrolysis. The nanocellulosic materials were deposited on a specially designed silicon wafer with grooves 227 nm in width, and a three-point bending test was applied to determine the elastic modulus using an AFM cantilever. The elastic moduli of single microfibrils prepared by TEMPO-oxidation and acid hydrolysis were 145.2 ± 31.3 and 150.7 ± 28.8 GPa, respectively. The result showed that the experimentally determined modulus of the highly crystalline tunicate microfibrils was in agreement with the elastic modulus of native cellulose crystals.