The layer-by-layer technique has been widely adopted for the fabrication of nanostructures with tailored properties. In this work, photoactive multilayer films consisting of alternating layers of chitosan and an azopolymer were developed by this method. Taking into account that pH is the factor controlling the charge of weak polyelectrolytes, the influence of this parameter on the structure and properties of the multilayer films was evaluated. Thus, different films were prepared by varying pH conditions as well as bilayer number. The morphology and properties of the films were analyzed by diverse advanced techniques, such as ultraviolet-visible spectroscopy, X-ray diffraction, and atomic force microscopy equipped with PeakForce QNM (Quantitative Nanomechanical Property Mapping). It was found that the thickness, roughness and elastic modulus of the developed multilayer films increased with the decrease of the chitosan solution pH and the increase of the bilayer number. Furthermore, induced birefringence measurements revealed that a higher level of photo-orientation was attained with the decrease of pH and the increase of bilayer number.