The present work focused on the synthesis of β-chitosan submicron particles (CSPs) from Todarodes pacificus using mechanochemical techniques. The gladius was submitted to a sequence of mechanical and chemical treatments to synthesize β-chitin (CT), which was further deacetylated to form spherical chitosan submicron particles with an average diameter of ⩽100 nm. The surface morphology of β-chitin and CSPs was observed using electron microscopy. The degree of deacetylation (DD%), evaluated from the absorbance peak of a Fourier Transform Infrared (FTIR) spectrum, was 80 ± 2.5%. Physicochemical characterization exhibited good crystallinity, positive zeta potential and low molecular weight, as well as reduced ash content and high water-binding capacity. CSPs exhibit significant antimicrobial properties toward all tested pathogenic bacterial and fungal microorganisms. Antioxidant analysis revealed high reducing power and excellent scavenging and chelating ability. Hence, CSPs synthesized from gladius of Todarodes pacificus using mechanochemical techniques are promising candidates for biomedical applications.