Biosurfactant was obtained from Nocardiopsis VITSISB and it was used in cosmetic formulation of toothpaste, replacing Sodium Lauryl Sulphate which is normally used in commercial toothpaste as a surfactant. This biosurfactant toothpaste was qualitatively analyzed by several tests like Spreadability Test, Foaming ability Test, Abrasiveness Test, Brine shrimp Hatchability Test and cleaning ability test. The results indicate that biosurfactant are more efficient and less toxic surfactant compare with chemical surfactant. In conclusion, the biosurfactant obtained from actinobacteria can act as good ingredient in place of chemical surfactant for toothpaste formulation. So with the advantage of environmental compatibility, in future the biosurfactant may be used in other cosmetic formulation like Shampoo formulation, face wash formulation etc. as an alternative for the chemical surfactant.