Mulberry trees are distributed throughout Pakistan. Besides its use in forage and food for animals, it is also used as herbal medicine. This study is carried out to determine phenolic acids profile, sugar content, and the antioxidant activity of the leaves and fruits of three mulberry species (Morus alba L., Morus nigra L. and Morus laevigata W.) grown in Pakistan. Dried fruits and leaves of three mulberry plant species were extracted by three different methods. The extracts were analysed by HPLC with diode array detection for phenolic acids. Antioxidant activity and sugar content were determined using spectrophotometric assays. The structure of chlorogenic acid was confirmed by LC-MS/MS analysis. The major phenolic acids were identified as gallic, protocatechuic, p-hydroxybenzoic, vanillic, chlorogenic, syringic, p-coumaric, ferulic and m-coumaric acids. Chlorogenic acid was the prominent phenolic acid in all leaves samples collected from three mulberry species and optimum extraction of chlorogenic acid was achieved using 80% aqueous methanol with ultrasonic-assisted extraction. Total phenolic compounds as determined by Folin-Ciocalteu assay were in the range of 3.89 to 11.79 mmol/ 100 g gallic acid equivalent, whereas antioxidant activities by DPPH assay ranged from 22.85–76.88 μmol/100 g quercetin equivalent. The concentration of sugars in different samples ranged between 36.56-82.15 mmol/ 100 g sucrose equivalent. This is the first report on identification of phenolic acids and antioxidant activity in the extracts of three cultivars of mulberry plants grown in Pakistan. This information will be of considerable value to the commercial producers of mulberry trees in the country.