The electrospinning technology opens up enormous possibilities for the implementation of bio-based materials and food hydrocolloids in numerous applications. In this context, chitosan is a sustainable, biocompatible, biodegradable, antimicrobial and non-toxic polysaccharide of great relevance in many fields of application. Because of its abundance in nature and excellent biocompatibility, the cationic polysaccharide chitosan is a very promising polymer for producing functional nanofibers. Although the material has good physicochemical properties, the electrospinning of the polymer is far from easy. In the current study, the effect of an unprecedented number of parameters (including solvent nature, polymer origin, molecular weight and spinning conditions) on morphology is reported. The work also aims to ascertain the antimicrobial properties of the generated biofibers of chitosan and relate them to its chemical structure. Finally, a new route is provided to generate chitosan based nanoporous structures starting from blends of chitosan and polylactic acid.