Unlike many biopolymers, α-1,3-glucan (α-1,3-GLU) is water-insoluble, making it a promising candidate for the production of moisture-resistant films with applications in biodegradable packaging, biomedicine, and cosmetics. This study aimed to characterize the structural, physicochemical (water affinity, optical, mechanical), and biodegradation properties of a film made from α-1,3-GLU extracted from Laetiporus sulphureus. The film was fabricated through alkaline dissolution, casting, drying, washing to remove residual NaOH, and re-plasticization with a glycerol solution. FTIR and Raman spectroscopy confirmed the polysaccharide nature of the film, with predominant α-glycosidic linkages. The film exhibited a semi-crystalline structure and high opacity due to surface roughness resulting from polymer coagulation. Owing to re-plasticization, the film showed a high moisture content (~47%), high water solubility (81.95% after 24 h), and weak mechanical properties (tensile strength = 1.28 MPa, elongation at break ≈ 10%). Its water vapor permeability (53.69 g mm m−2 d−1 kPa−1) was comparable to other glycerol-plasticized polysaccharide films reported in the literature. The film supported the adhesion of soil microorganisms and target bacteria and was susceptible to degradation by Trichoderma harzianum and endo- and exo-α-1,3-glucanases, indicating its biodegradability. The limitations in its mechanical strength and excessive hydration indicate the need for improvements in the composition and methods of producing α-1,3-GLU films.