Context: Monochoria vaginalis and Monochoria hastata belonging to Pontederiaceae are edible aquatic herbs commonly used by ethnic communities of India for treating various afflictions. Objective: The main objective of the study is to propose a simple and a rapid bioautographic fingerprinting profile using high-performance thin-layer chromatography (HPTLC) with gradient elution for screening the phytochemicals for the antioxidant property. Quantification of stigmasterol is carried in selected parts of Monochoria genus. Materials and Methods: Stigmasterol content of the three different parts of the species were quantified and validated by HPLTC. Fingerprint analysis was carried out using HPTLC-automated multiple development 2 (HPTLC-AMD2)-based gradient elution technique. Bioautography was done using 1-diphenyl-2-picrylhydrazyl (DPPH) solution to check the antioxidant property. Phytochemicals such as phenols, flavonoids, sterols, and saponins were estimated using 96-well plate and antioxidant potential were confirmed by DPPH, hydroxyl and nitric oxide scavenging activity. Results: The developed HPTLC-AMD2 method showed clear separations resulting in sharp, intense peaks. Phytoconstituents were determined for the first time in the Monochoria species. The results indicate that both species are rich in various bioactive contents with potent antioxidant potential. Stigmasterol was found to be present in all the selected parts with varying concentration. Conclusion: This study reveals that Monochoria species are rich in phytoconstituents with potent antioxidant activity and the developed method is efficient, simple, rapid, and reliable for separating the phytoconstituents of Monochoria providing a passport data of extracts. A positive, significant linear relationship between antioxidant activity and total phenol content and total flavonoid content showed that phenolic compounds and flavonoids were the dominant antioxidant components present in the extracts.