Quercus rotundifolia bark was studied regarding anatomical, chemical, and antioxidant properties from trees in two sites in southern Portugal and are here reported for the first time. The general structure and anatomy of Q. rotundifolia bark showed a rhytidome with sequential undulated and anastomosed periderms with a small proportion of cork, while the phloem included broad rays with strong cell sclerification, groups of sclereids with embed large prismatic crystals, and abundant druses in parenchyma cells. The mean chemical composition was 15.5% ash, 1.6% dichloromethane extractives, 6.4% ethanol and 9.3% water extractives, 3.0% suberin, 30.5% total lignin, and 33.8% carbohydrates. Carbohydrates included mainly glucose (50.7% of total monomers) and xylose (23.8%), with uronic (3.0%) and acetic acids (1.0%). Suberin was mainly composed of ω-hydroxyacids (48.0% of all compounds) and α,ω-diacids (19.5%). The main compounds found in the lipophilic extracts were triterpenes (43.6%–56.2% of all compounds) and alkanoic acids (32.7%–41.7%). Phenolic content was high especially in the ethanol extracts, ranging from 219.5–572.9 mg GAE/g extract and comprising 162.5–247.5 CE/g extract of flavonoids and 41.2–294.1 CE/g extract of condensed tannins. The extracts revealed very good antioxidant properties with IC50 values of 4.4 µg ethanol extract/mL and 4.7 µg water extract/mL. Similar anatomical, chemical, and antioxidant characteristics were found in the bark from both sites. The high phenolic content and excellent antioxidant characteristics of polar extracts showed holm oak barks to be a promising natural source of antioxidants with possible use in industry and pharmaceutical/medical areas.