Mulberry fruits are known as rich sources of anthocyanins and are consumed in syrup form after the addition of sugar and acid; however, there is little information on the anthocyanin composition and antioxidant activity of mulberries of different cultivars and their changes during processing. To address this, the antioxidant activity and anthocyanin composition of 12 cultivar mulberry fruit cultivars were investigated by high-performance liquid chromatography and ultra-high-performance liquid chromatography coupled with electrospray ionization/quadrupole time-of-flight. Additionally, different quantities of citric acid were used to evaluate antioxidant activities and anthocyanin composition of mulberry syrup. Sixteen anthocyanins were identified in mulberry fruits using accurate mass spectrometry. Several anthocyanins were tentatively identified for the first time in mulberry fruits and include: malvidin hexoside, cyanidin malonyl hexose hexoside, cyanidin pentoside, cyanidin malonyl hexoside, petunidin deoxyhexose hexoside, and cyanidin deoxyhexoside. The major anthocyanin in mulberries was cyanidin-3-O-glucoside, followed by cyanidin-3-O-rutinoside. Morus Alba L. Iksu showed the highest cyanidin-3-O-glucoside content (8.65 mg/g dry weight) among 12 mulberry fruit cultivars. As citric acid levels increased, mulberry syrup showed significantly higher antioxidant activity (p < 0.05).