In folklore medicine, Vitex negundo leaf is used to treat inflammation, pain, runny nose, itching, tumor etc. Different fractions of crude methanol extract of leaf of Vitex negundo Linn. were tested to evaluate antioxidant, analgesic, thrombolytic, and antibacterial activities. Crude methanol (MeOH) extract and its fractions i.e. petroleum ether (PEFS), CHCl3 (CFS), CCl4 (CTFS), aqueous (AQFS) were subjected to antioxidant activity by DPPH free radical scavenging assay. Analgesic activity was investigated by acetic acid induced writhing method at the dose of 250 and 500mg/kg body weight. Furthermore, thrombolytic activity was evaluated by clot lysis method and antibacterial activity by disk diffusion assay. In DPPH radical scavenging assay, crude extract presented the highest (IC50 value 25µg/ml) radical scavenging activity. Different fractions of extract at the tested doses showed significant (P<0.05) analgesic activity that was comparable to standard diclofenac sodium (80.32 % at 25mg/kg). Also different fractions revealed different range of antibacterial activity against different species of bacteria at different doses. Different fractions of V. negundo leaf disclosed potent antioxidant, thrombolytic, analgesic and antibacterial activities that support the ethnomedical uses of the plant.