The impact of Ca2+ counterions on the adsorption at the air-water interface and self-assembly in aqueous solution of the rhamnolipid biosurfactant and its mixture with the anionic surfactant sodium dodecylbenzenesulfonate, LAS, has been studied using neutron reflectometry and small-angle neutron scattering. The results illustrate how rhamnolipids are calcium tolerant and how their blending with conventional anionic surfactants improves the calcium tolerance of the anionic surfactant. Ca2+ has relatively little effect upon the adsorption and self-assembly of the monorhamnose, R1, and dirhamnose, R2, rhamnolipids, even at high pH, due to their predominantly nonionic nature. For R1/R2 mixtures the addition of Ca2+ has little impact upon the adsorbed amount or the surface composition. For R2/LAS mixtures the addition of Ca2+ results in an increased adsorption and a surface slightly richer in R2. The weak binding of Ca2+ to R1 and R2 does result in a change to the degree of ionization of the micelles and especially for mixed R1/R2 micelles at RI-rich solution compositions. The stronger binding of Ca2+ to LAS results in the addition of Ca2+ having a much greater impact on the self-assembly of R1/LAS and R2/LAS mixtures. For R1/LAS mixtures the addition of Ca2+ promotes the formation of more planar structures, even at low surfactant concentrations where in the absence of Ca2+ mixed globular micelle formation dominates. In R2/LAS mixtures, where there is a greater contrast between the high and low preferred curvatures associated with R2 and LAS, the addition of Ca2+ results in a more complex evolution in micellar aggregation and the degree of ionization of the micelles. This results in variations in Ca2+ binding that promotes micellar structures in which a spatial segregation of the two surfactant components within the micelle occurs.