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Mushrooms are edible fungi containing valuable nutrients. They provide attractive bio-active properties, which have confirmed anti-oxidants, anti-aging, and anti-inflammatory properties. Mushrooms possess abundant natural polymers affecting skin hydration and acting as moisturizers supporting skin barrier function. In this study, cloud ear mushroom (Auricularia polytricha) water extract (CW) was produced as a natural polymer to evaluate a new film-forming spray (FFS) containing CW to increase skin hydration and protect transepidermal water loss. CW contained polysaccharides as 748.2 ± 0.02 mg glucose/g extract. CW significantly inhibited the secretion of IL-6 and TNF-α and enhanced skin hydration by increasing aquaporin-3 (AQP3) and filaggrin (FLG) in HaCaT cells. The FFS was formulated using CW, sodium polystyrene sulfonate, and glycerin. The selected formulation contained brown Agaricus bisporus (BE-FFS) evaluated physical appearance, spray angle, spray pattern, and in vitro skin permeation. The BE-FFS has a transparent thin film with suitable occlusive properties, drying time, and physical appearance. Afterward, in vitro skin permeation and human hydration property studies presented the long-lasting effects and provided safety and hydration potential after 4 weeks of use. Overall, all results indicate that the BE-FFS is a natural film-forming spray for skin hydration improvement. [ABSTRACT FROM AUTHOR]