To examine the usage of biosurfactants as potential alternatives to chemosynthetic surfactants in controlling bubble behavior in the flotation process, a highspeed photographic method was employed to measure the motion of single bubbles and the size distribution of bubbles in the presence of biosurfactants in a laboratory scale flotation column. Deionized water, rhamnolipid, tea saponin and t-C8phenolethoxylateEO9 were used for making various surfactant solutions. Bubble trajectory, dimensions, velocity and size distribution were then determined from the recorded frames using the image analysis software. The results show that similar to chemosynthetic surfactants, the addition of biosurfactants has significant effects on bubble motion and size distribution. The addition of a small amount of tea saponin can significantly dampen bubble deformation, slow down terminal velocity, stabilize bubble trajectory, reduce bubble size and increase the specific surface area of bubbles due to the Marangoni effect. In addition, the biosurfactant effect on bubble behavior has been also found to depend on their type and concentration. The effect of tea saponin, fairly close to C8phenoleth-oxylateEO9, is stronger than rhamnolipid. The findings in the present study suggest that such biosurfactant as tea saponin may be good substitutes of chemosynthetic surfactants to control bubble behavior in flotation operation.