Aim: The study herein was performed in view of prospecting 15 species and one variety of Barleria in comparison to Adathoda vasica for phytochemical composition, and antioxidant activities. Materials and Methods: In addition to this, the study also quantifies two anti-bronchial (vasicine, vasicinone) and one anti-cancer (betulin) compounds using reversed-phase high-performance liquid chromatography analysis. Results: Samples showed betulin ranging from 7.700 to 73.447 mg/g, vasicine from 0.092 to 3.710 mg/g, and vasicinone from 0.005 to 2.752 mg/g. Principal component analysis and hierarchical cluster analysis results signified the division of seventeen samples tested, into two major clades (based on a similarity range 57.83–96.60%). Conclusion: Both phytochemical analysis and antioxidant activities showed variation in the samples tested. Barleria grandiflora had higher content and better activities among the species.