To elucidate the effects of cultivar and harvest month on the phenolic content and antioxidant activity of mulberry leaves, four major phenolics, including chlorogenic acid (ChA), benzoic acid (BeA), rutin (Rut) and astragalin (Ast), were quantified using an HPLC-UV method. Leaves from six mulberry cultivars, collected from April to October, were analyzed. The antioxidant activity of mulberry leaves was assessed by ferric reducing antioxidant power (FRAP), hydroxyl radical scavenging activity (HSA) and superoxide radical scavenging activity (SSA) assays. The results showed that the total values of the four phenolic compounds ranged from 2.3 dry weight (DW) to 4.2 mg/g DW, with ChA being the major compound. The mean total phenol (TP) content of the six cultivars ranged from 30.4 equivalents (GAE) mg/g DW to 44.7 GAE mg/g DW. Mulberry leaves harvested in May had the highest TP content. Moreover, the antioxidant activities of mulberry leaves harvested from April to October differed noticeably. In general, Kq 10 and May were considered to be a better cultivar and harvest month concerning phenolic content and antioxidant activity, respectively.