The content of chemical constituents in Eugenia uniflora leaf extracts correlates positively with biological activities. The experimental objective was to carry out the phytochemical screening and purification of the major polyphenols from the leaves of E. uniflora. In addition, the anti-Candida activity of the hydroalcoholic extract, fraction, subfractions and polyphenols purified were evaluated. After partitioning of the extract with ethyl acetate, the fractions were chromatographed on Sephadex^® LH-20 gel followed by RP-flash chromatography and monitored by TLC and RP-HPLC. The samples were characterized by mass spectrometry (LC-ESI-QTOF-MS²) and subjected to the microdilution method in 96-well plates against strains of C. albicans, C. auris, and C. glabrata. Myricitrin (93.89%; w/w; m/z 463.0876), gallic acid (99.9%; w/w; m/z 169.0142), and ellagic acid (94.2%; w/w; m/z 300.9988) were recovered. The polyphenolic fraction (62.67% (w/w) myricitrin) and the ellagic fraction (67.86% (w/w) ellagic acid) showed the best antifungal performance (MIC between 62.50 and 500 μg/mL), suggesting an association between the majority constituents and the antifungal response of E. uniflora derivatives. However, there is a clear dependence on the presence of the complex chemical mixture. In conclusion, chromatographic strategies were effectively employed to recover the major polyphenols from the leaves of the species.