Background: Rostellularia diffusa is an unexplored medicinal plant used as brain tonic in traditional medicine system. Objective: This study was designed to investigate the antioxidant and anti-stress potential of R. diffusa by experimental animal models. Materials and Methods: The extracts of R. diffusa were subjected to preliminary phytochemical screening and high performance thin layer chromatography (HPTLC) finger printing analysis. The antioxidant potential of the extracts was found by different in vitro models. The anti-stress activity was investigated by using acetic acid induced writhing test, swimming endurance test, and restraint stress in experimental mice. Serum parameters such as glucose, triglyceride and cholesterol, oxidative stress parameter thiobarbituric acid reactive substance, antioxidant parameters such as reduced glutathione, superoxide dismutase and catalase and organ weights were evaluated after restraint stress in mice. Diazepam was used as reference standard to compare the anti-stress activity of plant extract. Results: High performance thin layer chromatography finger printing analysis revealed the presence of flavone compounds in both extracts. The extracts also showed good antioxidant property in different in vitro antioxidant models. Administration of extracts of R. diffusa decreased the number of wriths and immobility time when compared with control group in acetic acid-induced writhing test and swimming endurance test respectively in experimental mice. They also suppressed the restraint stress-induced alterations in serum parameters, oxidative stress, and antioxidant parameters in brain and also restored the organ weights in normal level. Conclusion: From these results, it has been concluded that the potential anti-stress activity of R. diffusa is through its adaptogenic and antioxidant properties.