Ultrafast optical nonlinearity in chitosan (CS) films doped with multi-walled boron nitride nanotubes (MWBN) has been investigated using 800 nm, 100 fs laser pulses, employing the open aperture Z-scan technique. Two-photon absorption coefficients (β) of CS–MWBN films have been measured at 800 nm by Z-scan. While chitosan with 0.01% MWBN doping gives a β value of 0.28×10−13 m/W, 1% doping results in a higher β value of 1.43×10−13 m/W, showing nonlinearity enhancement by a factor of 5. These nonlinearity coefficients are comparable to those reported for silver nanoclusters in glass matrix and Pt-PVA nanocomposites, indicating potential photonic applications for MWBN doped chitosan films. Characterization of the synthesized films using Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) reveals significant interactions between the NH and CO groups of chitosan with boron nitride.