The bark of Eucalyptus globulus is a promising source of polyphenolic compounds that could be extracted employing polar solvents. Extraction experiments were planned according to the Box−Behnken design to evaluate the effect of temperature and time of extraction and the ethanol/water ratio on the dependent variables total phenolic content (TPC; as gallic acid equivalents, GAE), antioxidant activity (AA; as millimoles of ascorbic acid equivalents, AAE), proanthocyanidins (Pac; as mimosa extract equivalents, MEE), Stiasny number (SN), and coextracted total carbohydrates (TC). Response surface models were developed, and statistical analysis of variance was performed. A maximum of TPC of 32% (w GAE /w extract ) was achieved for 264 min, 82.5°C, and 52% ethanol corresponding to about 2% of the bark weight. These conditions are close to those leading to maximum values of compounds with AA (2.1 mmol AAE /g extract ) and Pac 14% (w MEE /w extract ). TC in the extract and the SN predicted were 22.7% (w TC /w extract ) and 37, respectively. Preliminary studies concerning the biological activity of some extracts have demonstrated their differential capacity to reduce human breast cancer cell proliferation.