This study aims to investigate the effects of different fermentation strains on the active components of the fermentation broth of Sequoia sempervirens stem, and to evaluate its potential in enhancing skin barrier function and anti-inflammatory activity. The bark of Sequoia sempervirens stem was used as a starting material to determine the content of active ingredients after fermentation with different strains. The optimal strain combination was selected to ferment the resulting liquid, which was then subjected to cell toxicity tests, cell scratch assays, hyaluronidase activity inhibition, inhibition of inflammatory factor release, free radical scavenging, and anti-zebrafish tail fin shrinkage experiments to validate its efficacy in enhancing skin barrier function and anti-inflammatory effect.The results showed that the compound fermentation system of Saccharomyces cerevisiae 1-1 and Bacillus subtilis 2-1 significantly increased the content of active ingredients in the fermentation broth. This fermentation broth exhibited remarkable hyaluronidase inhibitory activity in vitro, with an IC₅₀ value of 9.89 mg/mL and showed strong scavenging ability against DPPH free radicals, superoxide anions free radicals, and hydroxyl free radicals. Additionally, the cellular experimental results indicated that low to moderate concentrations of the fermentation broth had no obvious cytotoxic effects on HaCaT cells, while 10 mg/mL significantly promoted the wound healing of HaCaT cells. Further study revealed that the fermentation broth with concentration of 10 mg/mL could effectively inhibit the levels of Tumor necrosis factor-α and Interleukin-6 secreted by macrophages stimulated with lipopolysaccharide Finally, the anti-photoaging effect of the fermentation broth under UV radiation was verified by the zebrafish tail fin wrinkle experiment. This study proposes an efficient method for preparing active ingredients from the bark of Sequoia sempervirens stem and demonstrates the potential value of the fermentation broth in enhancing skin barrier function and anti-inflammatory activity, providing a new research direction for the application of Sequoia sempervirens in skincare, repair, and anti-aging.