The objective of this work was the recovery of chitosan by ultrasound-assisted extraction (UAE) from white shrimp (Litopenaeus vannamei) chitin, and the physicochemical and structural characterization of the obtained biopolymer, as well as its antimicrobial effect on Pythium ultimum growth. A 2³ factorial design was used to evaluate chitosan extraction conditions. Instrumental analysis techniques for chitosan characterization and radial growth inhibition, as an antifungal activity test, were performed. The ultrasonically extracted chitosan (UC) reached a yield of 86.96% with 100% solubility, a degree of deacetylation (DDA) >78%, molecular weight (M_W) of 3.928 × 10⁵ g mol⁻¹, and a crystallinity index (Icr) of 87%, calculated through nuclear magnetic resonance (¹H NMR) and Fourier transform infrared spectroscopy (FTIR), size exclusion chromatography (SEC), and X-ray diffraction (XRD), respectively. The inhibitory activity of the chitosan was evaluated against the oomycete Pythium ultimum, observing a 93% radial inhibition over 24 h. UAE proved to be an excellent alternative to the conventional deacetylation, reducing reaction time and obtaining a UC with higher M_W and (Icr) than the commercial one, which could potentiate its applications.