For the first time, grape seed extracts (GSEs), obtained from wine and table cultivars of Vitis vinifera L., cultured in experimental fields of Lazio and Puglia regions of Italy and grown in different agronomic conditions, have been tested on 43 Candida species strains. We demonstrated a significant correlation between the content of the flavan-3-ols in GSEs extracts, with a polymerization degree ≥ 4, and anti-Candida activity. Moreover, we demonstrated that GSEs, obtained from plants cultured with reduced irrigation, showed a content of polymeric flavan-3-ols >250 mg/g with geometric mean MIC values between 5.7 and 20.2 mg/L against Candida albicans reference strains. GSE, showing 573 mg/g of polymeric flavan-3-ols, has been tested in an experimental murine model of vaginal candidiasis by using noninvasive in vivo imaging technique. The results pointed out a significant inhibition of Candida albicans load 5 days after challenge. These findings indicate that GSEs with high content of polymeric flavan-3-ols can be used in mucosal infection as vaginal candidiasis.