The crude methanol extract of leaf of Tiliacora acuminata Miers as well as organic solvent (n-hexane, carbon tetrachloride and chloroform) fractions and methanolic fraction (aqueous) were subjected to screening for total phenolic content, antioxidant, cytotoxic, thrombolytic and antimicrobial activity. In total phenolic content analysis, the n-hexane soluble fraction of leaf was found to contain highest amount of phenolic content (TPC, 132.18 mg/gm of dry weight of extract, expressed as gallic acid equivalents). In the DPPH assay, the n-hexane soluble fraction of leaf displayed the highest free radical scavenging activity with IC50 value 64.54 μg/ml as compared to 27.5 μg/ml produced by butylated hydroxyl toluene.  A positive correlation was evident between total phenolic content and free radical scavenging activity of T. acuminata having correlation coefficient (R2) of 0.91. In cytotoxicity screening, the crude methanol extract of leaf demonstrated strong cytotoxic activity with LC50 value of 2.40 μg/ml as compared to 0.451 μg/ml produced by vincristine sulphate. During assay for thrombolytic activity, the crude methanol extract revealed 32.5% lysis of clot while standard streptokinase and water, used as positive and negative controls, demonstrated 70.4% and 3.62% lysis of clot, respectively. In antimicrobial assay by disc diffusion method, all the samples exhibited moderate to significant antimicrobial activity (zone of inhibition = 10.0-20.0 mm) against all the test organisms. Among all the samples, the carbon tetrachloride soluble fraction displayed strong antimicrobial activity against Escherichia coli (20.0 mm).