Oxidative stress (OS) is involved in pathophysiological events of different diseases. Antioxidants mitigate and suppress the adverse effect of OS in the body. Due to lesser side effects and cost effectiveness natural antioxidant is a choice of people nowadays. The present study was designed to characterize/analyze phytochemicals and antioxidant potential in Parthenium hysterophorus (Asteraceae) stem (PHS) sequentially extracted fractions. Chemical characterization of PHS samples was carried out using Gas Chromatography-Mass Spectrometry (GC-MS) and Spectrophotometric techniques. Comparative antioxidant potential in PHS fractions was studied using in vitro antioxidant models. Thin layer chromatography (TLC) was performed to show a differential occurrence of phytochemicals in test fractions and related radical scavenging potential. PHS fractions showed differential presence of compounds. Appreciable radical scavenging, reducing power, and metal ion chelation potential was found in PHS fractions. In conclusion, P. hysterophorus stem may be considered a good source of natural antioxidant compounds.