Alternanthera philoxeroides (Mart.) is a tropical weed commonly known as alligator weed. It grows rapidly within a small span of time and easily available all over the world. The objective of this work was to isolate and characterise the major phenolic components present in the methanol-soluble fraction (fraction X) of A. philoxeroides leaves and to explore the biological activity (antioxidant, α-glucosidase inhibition and antimicrobial) of the fraction in in vitro system. Chromatographic (HPLC) and spectroscopic (MALDI-TOF, 1H NMR) techniques were used to purify and characterise the phenolics present in fraction X. Five major phenolics (kaempferol, ferulic acid, salicylic acid, syringic acid and chlorogenic acid) were found in fraction X. The fraction showed anti-oxidant property, dose-dependent inhibition of α-glucosidase activity and anti-microbial activity. Hence fraction X from the weed has therapeutic potential in pathophysiological condition. © 2014 Taylor & Francis.