The present investigation was focused on the antioxidant activity of the methanolic extract of Rotula aquatica, a medicinal shrub trailing over gravel in stream beds. The extract exhibited significant (p<0.05) increase in the antioxidant activity with increase in concentration. The extract showed 91.89% DPPH scavenging activity and 74.35% inhibition of nitric oxide at a concentration of 128 μg/mL. Superoxide scavenging activity indicated that 38 μg/mL of extract could inhibit 50% of superoxides. FRAP assay and total reducing potential also signifies the antioxidant potential of the plant extract. The study demonstrated a statistical correlation (r>0.95) between phenol content and antioxidant activity. Overall, the work validates the ethnobotanical usage of the plant and its potential for application in pharmaceutical and food industries.