Bubble hydrodynamics is fundamental to the performance of the flotation process widely used in the separation industry. To compare the effect of biosurfactants and chemical synthetic surfactants on bubble hydrodynamics in the flotation process, the motion of a single bubble and the size distribution of bubble swarms in various surfactants (rhamnolipid, tea saponin and Triton X-100) solutions were observed directly using a high-speed video camera in a laboratory scale flotation column. Bubble trajectory, dimensions, velocity and size distribution were then determined through image analysis. The results indicated that the addition of biosurfactants had the same significant effects on bubble motion and size distribution as chemosynthetic surfactants. The biosurfactant effect on bubble behavior was also found to depend on their type and concentration. In general, the effect of tea saponin was stronger than another biosurfactant (rhamnolipid) used in the present study. The present findings implied that some biosurfactants like tea saponin can replace chemosynthetic surfactants in controlling bubble behavior in flotation operation. This will contribute to promoting the use of green environmentally friendly flotation agents in the separation industry.