Background: Obesity is a metabolic disorder that has reached pandemic proportions worldwide. Phytol, a diterpene isoprenoid alcohol found as an integral part of chlorophyll of plants, algae, and guts of ruminant animals, is proven for its lipid-lowering activity. This compound is largely present in the Indian aquatic medicinal plant, Hydrilla verticillata. The research is focused to screen the anti-adipogenesis activity of H. verticillata extract on 3T3 L1 cell lines. Materials and Methods: The ethanolic extract of H. verticillata was prepared and characterized by gas chromatography–mass spectroscopy and Fourier transform infrared spectroscopy analysis. In vitro cytotoxic effect of H. verticillata extract on 3T3 L1 cell line was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test. The ethanolic solvent extract of this plant is screened for anti-adipogenesis activity by performing in vitro pancreatic lipase inhibition and Oil Red O staining assay. Results: Phytochemical analysis and characterization of the extract showed the presence of a large amount of phytol in H. verticillata. In vitro pancreatic assay of the crude extract showed maximum lipase inhibition activity at a minimal concentration of 125 μg/mL, similar to the standard anti-obesity drug, orlistat. The half-maximal inhibitory concentration (IC50) of the extract on 3T3 L1 cell line was 840.91 μg/mL. Cells treated with one-third IC50showed minimal lipid accumulation, which is determined by Oil Red O staining. Conclusion: This study confirms that the ethanolic extract of H. verticillata possesses anti-adipogenesis activity, which can be used to control obesity-related treatments.