Background: Blechnum orientale Linn. (Blechnaceae), a fern, is traditionally used in the treatment of various ailments, such as skin diseases, stomach pain, urinary bladder complaints, and also as a female contraceptive. Previously, we reported a strong radical scavenging activity, antibacterial activity and cytotoxicity against HT29 colon cancer cells by aqueous extract of B. orientale. Objective: In this study, we attempted to isolate and identify the active compound from the aqueous extract of B. orientale. Materials and Methods: Aqueous extract of B. orientale was subjected to repeated MCI gel chromatography, Sephadex-LH-20, Chromatorex C18 and semi-preparative high performance liquid chromatography and was characterized using nuclear magnetic resonance and electrospray ionization mass-spectrometry spectroscopic methods. Antioxidant activity was determined using 2, 2-diphenyl-1-picrylhydrazyl radical scavenging assay. Antibacterial assays were conducted using disc diffusion whereas the minimum inhibitory concentration (MIC) and minimum bactericidal concentration were determined using the broth microdilution assay. Cytotoxicity was assessed using thiazolylblue tetrazoliumbromide. Results: A polymeric proanthocyanidin consisting of 2-12 epicatechin extension units and epigallocathecin terminal units linked at C4-C8 was elucidated. Bioactivity studies showed strong radical scavenging activity (IC50 = 5.6 ± 0.1 µg/mL), antibacterial activity (MIC = 31.3-62.5 µg/mL) against five gram-positive bacteria and selective cytotoxicity against HT29 colon cancer cells (IC50 = 7.0 ± 0.3 µg/mL). Conclusion: According to our results, the proanthocyanidin of B. orientale demonstrated its potential as a natural source of antioxidant with antibacterial and anti-cancer properties.