An innovative one-step approach based on femtosecond laser ablation synthesis in aqueous solution is presented for the development of copper nanoparticle-chitosan (CuNP-CS) nano-antimicrobials. The influence of the CS concentration has been assessed, in order to tune the morphological and chemical features of the as-prepared nanomaterial. CuNP-CS hybrid nanocolloids have been characterized by TEM and XPS techniques. Ultrafine and almost monodisperse CuNPs are obtained at a CS concentration of 1 g/L.