Calafate fruits have a high content of phenolic compounds and an antioxidant activity up to four times higher than that of blueberries. The establishment of a calafate orchard and irrigation responses on fruit and soil characteristics have been scarcely studied. Therefore, the objective of this study was to evaluate the effect of water replenishment rate: 0%, 50%, 100% and 150% of reference evapotranspiration (ET0), on soil microbiological activity, plant physiological response, fruit yield and chemical composition in a calafate orchard. The results showed that irrigation at 50% ET0 presented significant increases in soil urease, dehydrogenase and acid phosphatase activity. Likewise, irrigation at 50% ET0 significantly increased stomatal conductance and plant chlorophyll index, which led to a significant increase in fruit yield being 60% higher compared to the other treatments. Despite the higher fruit yield, 50% ET0 irrigation had a similar level of total anthocyanins and ORAC antioxidant capacity as the 100% ET0 treatment. In contrast, 0% and 150% ET0 treatments showed a higher degree of stress and got higher values for total anthocyanins and fruit antioxidant capacity. Irrigation rates 50% ET0 increases fruit yield while maintaining fruit quality and optimizing water resources in commercial orchards of calafate.