Objective: The present study was focused to screen traditionally used Barleria prionitis for anticancer effects against various cell lines and antimicrobial effect against various pathogenic strains of bacteria and fungi.Methods: Extraction of Barleria prionitisleaves in ethanol was done by the Soxhlet method. After extraction, phytochemical estimation of these seven secondary metabolites like alkaloids, flavonoids, anthraquinones, saponins, terpenoids, tannins, and cardiac glycosides was done as per the protocols of Kokate. Minimum Inhibitory Concentration (MIC) effect of Barleria leaf ethanol (BLE) was done by the dilution method on five bacterial and five fungal strains. Further analysis (anticancer activity) was done with SRB (Sulphorhodamine B) assay. Statistical analysis of antimicrobial and anticancer activity was done by using MS Excel 2007 to±standard deviation and student t-test.Results: Barlerialeaf extract with ethanol is a non-polar solvent extract and considered as the best solvent to extract the maximum number of secondary metabolites like alkaloids, saponins, flavonoids, and tannins. BLE extract gave excellent MIC (Minimum Inhibitory Concentration) effects against pathogenic bacteria and pathogenic fungal strains. BLE had highly effective activity against Pseudomonas aeruginosa with 1.25 mg MIC, the OD value of the sample was 0.02±0.0005 (±SD) with 0.0211-0.0245 range. MIC against fungal strains had effective activity against Candida vaginitis with 6.25 mg, the OD value of the sample was 0.02±0.0003 (±SD) with 0.0213-0.0232 range. BLE extract had given more than 70% inhibition against breast cell lines (MCF-7) and 75.16% inhibition of DLD1 cell lines; it was near to Doxorubicin antibiotic (81%). Breast metastatic cell line (MDMAMB-468) was found 60% inhibited with BLE extract and there was a great difference in the results of Doxorubicin. Out of six experimented cell lines, BLE gave very good inhibition for two cell lines, i.e. Breast (MCF-7) and Colon cell lines (DLD-1).Conclusion: BLE extract had shown the best antimicrobial and antifungal effect, against Pseudomonas aeruginosa and Candida vaginitis respectively. BLE also showed an anticancer effect against Lung cell lines (A549), Breast cancer cell line (MCF-7), Breast metastatic cell line (MDMAMB-468), Colon cell line (DLD-1) and lung metastatic cell line (NCIH358) at a statistically significant level (p=<0.05). It did not give any significant results against the colon metastatic cell li...