[Background] Sugarcane smut disease is a main disease on sugarcane that causes considerable yield losses; rhamnolipids are biosurfactants that have been studied as an antifungal agent against various of plant diseases. [Objective] To evaluate the antifungal activity of rhamnolipids against S. scitamineum in vitro, exploring the preliminary antifungal mechanism of rhamnolipids against S. scitamineum. [Methods] The teliospores of sugarcane smut germination were employed to determine the antifungal of rhamnolipids. The method of mycelial growth rate and dry weight of mycelium were used to test in in vitro test of rhamnolipid. The effect of rhamnolipids on the membrane permeability of S. scitamineum that was assayed the change of electrical conductivity of the hyphae. [Results] The results showed that rhamnolipids could significantly inhibit the spore germination of S. scitamineum. 2.0 g/L rhamnolipids reduced the germination rate of teliospores by 45.03%. Rhamnolipids significantly inhibited the mycelial growth of the diploid, haploid-a and haploid-b strain of S. scitamineum. Rhamnolipids could increase the membrane permeability of cell membrane of S. scitamineum, compared with the control, the conductivity of diploid strain raised 9-fold at 2.0 g/L rhamnolipids after 5 min，the conductivity of haploid-a strain of S. scitamineum increased by 94.23% after 30 min at 2.0 g/L rhamnolipids. The conductivity of mycelium increased by 54.49% while haploid-b strain of S. scitamineum had been handled with 0.1 g/L rhamnolipids for 30 min. Mycelial conductivity raised with the increasing of the concentration of rhamnolipids. [Conclusion] Rhamnolipids showed a high antifungal activity against S. scitamineum, which could offer a new method for controlling sugarcane smut disease.