Abstract: This study investigated the antibacterial components and underlying mechanisms of action of gallic (Galla chinensis) extracts. The ethanol extract was separated into different fractionations by using silica gel column chromatography, followed by analysis via ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF MS). The mechanism underlying the inhibitory effect of gallic extract on Penicillium citrinum was elucidated by assessing of cell membrane integrity, oxidative stress, mitochondrial function, and cellular ultrastructure. The results demonstrated that the Fr.3 fraction exhibited an maximum inhibition zone against P. citrinum with the diameter of 17.85 mm. UPLC-QTOF MS analysis identified 12 bioactive compounds in Fr.3, including ellagic acid, gallic acid, and (-)-epigallocatechin, among others. Antifungal assays demonstrated that gallic acid and (-)-epigallocatechin inhibited P. citrinum growth by 53.99% and 45.75%, respectively, after 6 days of culture at 28 ℃. Gallic acid impaired cell membrane integrity, leading to a 78.80% increase in relative conductivity and a 60.16% increase in malondialdehyde content. Gallica acid also induced oxidative stress, resulting in elevated reactive oxygen species (ROS) levels, which subsequently disrupted intracellular redox homeostasis in P. citrinum cells. Additionally, gallic acid inhibited mitochondrial function of P. citrinum, resulting in a 76.88% decrease in ATPase activity, a decline in mitochondrial membrane potential, and a 94.22% increase in depolarization. Furthermore, the P. citrinum cell ultrastructure was compromised and plasmolysis was observed. These findings elucidate the multitarget inhibitory mechanism of G. chinensis at the molecular level and provide a theoretical framework for natural antifungal agent development.