The Young's modulus of multilayer films containing nanofibrillated cellulose (NFC) and polyethyleneimine (PEI) was determined using the strain-induced elastic buckling instability for mechanical measurements (SIEBIMM) technique. (1) Multilayer films were built up on polydimethylsiloxane substrates using electrostatic layer-by-layer assembly. At 50% relative humidity, SIEBIMM gave a constant Young's modulus of 1.5 ± 0.2 GPa for 35-75 nm thick films. Conversely, in vacuum, the Young's modulus was 10 times larger, at 17.2 ± 1.2 GPa. A slight decrease in buckling wavelength with increasing strain was observed by scanning electron microscopy with in situ compression, and above 10% strain, extensive cracking parallel to the compressive direction occurred. We conclude that whereas PEI acts as a "glue" to hold multiple layers of NFC together, it prevents full development of hydrogen bonding and specific fibril-fibril interactions, and at high humidity, its hygroscopic nature decreases the elastic modulus when compared with pure NFC films.