Polygonum capitatum (Buch.-Ham. ex D. Don) H. Gross is a perennial herb belonging to the Polygonaceae family, mainly distributed in Southwestern China. P. capitata is edible and boasts a high nutritional profile, and the entire plant serves medicinal purposes. According to the Guangxi Materia Medica, it has the functions of “relieving wind-dampness, enhancing blood circulation, and easing pain”. P. capitata is a major component of the traditional Chinese medicine formula, Re-Lin-Qing, which is widely used for treating urinary calculi (urolithiasis). However, its specific mechanisms of action remain unclear. In this study, we employed molecular biology, molecular dynamics, and cellular biology strategies to develop a rat experimental model of oxalate-induced urolithiasis and a cell model of oxalate-induced injury to study the active constituents and molecular mechanisms of P. capitata in inhibiting oxalate stone formation both in vivo and in vitro. The results show that in vivo, P. capitata significantly reduced the formation rate of oxalate stones in rats, decreased the levels of urinary oxalate, sodium, potassium, calcium, and magnesium, while increasing urine pH, urinary citrate levels, and urine excretion. Moreover, P. capitata significantly lowered serum creatinine levels in oxalate stone model rats and mitigated kidney damage. In vitro, treatment with P. capitata extract effectively alleviated oxalate-induced oxidative stress in HK-2 cells, reducing the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), while increasing ATP levels and inhibiting oxalate-induced apoptosis. LC-MS analysis of P. capitata components, in combination with transcriptomics, network pharmacology, and metabolomics sequencing, indicated that myricetin might be one of its potential bioactive compounds. Both P. capitata extract and its active compound, myricetin, significantly reversed the oxalate-induced decrease in phosphatidyqinositol-3 kinase (PI3K) and extracellular regulated protein kinases (ERK) levels and inhibited the overexpression of oxidative stress-related proteins. Further analysis using cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) experiments revealed that myricetin exhibits strong binding affinity to phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PI3KCA). Moreover, PI3K inhibitors reversed the protective effects of P. capitata and myricetin on oxalate-induced cell injury models. P. capitata exerts its anti-urolithiasis effects by alleviating oxidative stress through a multi-component, multi-target synergistic mechanism. Additionally, myricetin, one of the bioactive components of P. capitata, directly binds to PIK3CA, promoting ERK expression, upregulating nuclear factor E2-related factor-2 (NRF2) levels, and subsequently reducing oxidative stress, thereby inhibiting oxalate stone formation and alleviating kidney injury.