The growing request for more sustainable materials and environmentally friendly nanofabrication methods in the electronics field has recently driven the scientific community in the development of bio-derived materials as an alternative to conventional lithographic resists. In this work, we used chitosan, a biodegradable and biocompatible polysaccharide, as a green direct-write resist material for Atomic Force Microscopy-based nanolithography. Chitosan thin layers were obtained by spin coating and systematically characterized, in terms of thickness and surface roughness, demonstrating nanoscale smoothness and tunable film thickness. Three Pulse-Atomic Force Lithography (P-AFL) approaches, i.e., Constant Pulse, Gradient Pulse, and Raster Pulse AFL methods, were used to pattern nanostructures with constant-depth nanogrooves, variable-depth (2.5D) profile, and three-dimensional nanoholes on chitosan films. The results reveal high pattern fidelity, reproducibility, and tunability of feature dimensions as a function of applied force and scanning direction. Moreover, the RP-AFL technique enabled the fabrication of well-defined 3D nanostructures with depths matching the film thickness, which is a prerequisite for subsequent pattern transfer. This experimental work provided a first proof-of-concept to adopt chitosan as a more sustainable alternative with respect to conventional resists. Moreover, the results highlight P-AFL methods as a versatile and low-impact nanofabrication strategy, contributing to the development of greener micro- and nano-manufacturing technologies.