Black mulberry leaves teas (BMLTs) were prepared using boiled water and different steeping time (5 10, 20, 35 and 45 minutes). In order to establish the connection between steeping time and tea quality, total phenolics content (TPC), total flavonoids content TFC contents were measured, as well as two antioxidant assays (DPPH and reducing power assays), alone with antimicrobial and cytotoxic tests. The obtained results showed that TPC, TFC, IC50 and EC50 values increased with the increase in steeping time, while antimicrobial and cytotoxic activity exhibited different tendency. Based on the obtained results, the 10-minute steeping time was the optimum for tea preparation and this tea was used for the determination of polyphenolic profile using HPLC-MS technique. The results showed that the main compounds in BMLT were chlorogenic and caffeic acids with the contents of 7226.00 and 537.52 μg/g, respectively. [Projekat Ministarstva nauke Republike Srbije, br. TR31013]