Black mulberry fruit is processed to juice at significant scale in Turkey. The effect of industrial-scale juice production on black mulberry antioxidants was evaluated using samples collected from the main steps of processing; including the selection of fruits, washing, mechanical milling, mashing, cold pressing, pasteurization, and filling-packing. Two major anthocyanins (cyanidin-3-glucoside and cyanidin-3-rutinoside), two phenolic acids (3- and caffeoylquinic acid) and 3 flavonols (rutin, quercetin-3-glucoside, and quercetin-malonyl-glucoside) were identified using LC–QTOF–MS and were quantified using HPLC. Approximately, 60–70% of the fruit anthocyanins were retained in the final juice, which also contained high levels of caffeoylquinic acids, relative to the fruit. Mashing and pressing were the steps which were effective for the recovery of fruit polyphenolics into the juice fraction. Moreover, an in vitro gastrointestinal digestion model, applied to determine the effect of processing on the bioavailability of mulberry antioxidants, indicated a higher anthocyanin bioavailability for the fruit matrix than for the juice matrix.