Background: The root-bark of the medicinally important plant Zanthoxylum budrunga (ZBRB) brooks a variety of uses in ethnobotanical and ethnomedicinal practice in Bangladesh and thus demands biological investigation to reveal its therapeutic potentiality. So, the present study was perpetrated to explore antioxidant, analgesic, antidiarrhoeal, and a cytotoxic activity of ethanolic root-bark extract of Zanthoxylum budrunga and was also to quantify the major bioactive polyphenolic constituents by HPLC analysis. Methods: Total phenolic content was measured spectrophotometrically by using Folin Chiocalteu's reagent while in vitro antioxidant activity was determined by means of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay and reducing power assay. HPLC analysis was performed to identify and quantify the major bioactive polyphenolic constituents present in the extract. Acetic acid induced writhing test and hot-plate test were conducted to evaluate the analgesic activity of the crude extract. On the other hand, in vivo antidiarrheal potentiality was investigated by using experimentally castor oil induced diarrhoea in mice and brine shrimp lethality bio-assay was implemented to check the cytotoxic potentiality of the crude extract. Results: Zanthoxylum budrunga showed DPPH scavenging activity (IC 50 54.27 μg/mL), while the total phenolic content was 647.91 mg GAE/100 g of extract. ZBRB also showed concentration dependent ferric reducing power activity. At the doses of 250 and 500 mg/kg, ZBRB exhibited statistically significant (P < 0.001) inhibition of writhing in test mice (64.58 and 77.78%, respectively). In hot-plate test, ZBRB, at the above dose levels, significantly (P < 0.001) prolonged pain threshold with response time of 5.80 and 6.81 s, respectively. In castor oil induced diarrhoeal episode in mice, ZBRB exhibited 66.56% and 83.39% inhibition of defecation at the doses of 250 and 500 mg/kg, respectively (P < 0.001). The LC 50 (Median lethal concentration) value of ZBRB in brine shrimp lethality bioassay was found to be 21. 84 μg/mL. In the HPLC analysis, (+)-catechin, caffeic acid and quercetin were detected with the concentration of 17. 94 mg/100, 3.72 mg/100 and 11.95 mg/100 g of ethanolic extract of ZBRB, respectively. Conclusion: The results rationalize the uses of the plant in traditional medicine for diarrhoeal as well as pain management. Catechin, caffeic acid, and other phenolics constituents might have some function in the observed activity.