Biosurfactants are increasingly being considered as sources for bioremediation of toxic heavy metals due to their metal complexing abilities. Rhamnolipids are one group of glycolipid biosurfactants produced by Pseudomonas aeruginosa that have been shown to have potential for toxic heavy metal bioremediation. The interfacial chemistry of rhamnolipids at the air-water interface can affect the bioremediation process. Thus, the interfacial chemistry of rhamnolipids is being investigated in Langmuir films at the air-water interface of a Langmuir-Blodgett trough through the characterization of surface pressure-area isotherms under varying sub-phase conditions. Studies investigating the effects of sub-phase ionic strength and presence of the metal cations Al³⁺, La³⁺, Cd²⁺, and Pb²⁺ at different concentrations and pH are reported here. These variations in sub-phase chemistry affect the interfacial packing and molecular orientation of rhamnolipids at the air-water interface as indicated by the surface tension values and average interfacial molecular area.