Tertiary recovery is directly dependent on the alteration in wettability and interfacial tension (IFT), hence releasing the trapped oil from rock pore spaces. Bio-surfactant water flooding to mobilise residual oil in reservoirs is a new and developing prospect that can be used more often in future due to its environmentally friendly nature and economic advantages. In this work, the impact of rhamnolipids as water soluble bio-surfactant solutions on the interfacial activities of saline water and the wettability of carbonate rock are studied at elevated temperature. The effectiveness of the bio-surfactant as a rock wettability modifier is analysed in the presence of different salinities, in particular SO42− ions. The reason for the focus on SO42− is its high affinity towards calcite surfaces, and hence its ability to intervene strongly on bio-surfactant performance. To achieve the objectives of this study, the oil-wet calcite samples at elevated temperature were put through a washing process that included bio-surfactant solutions in seawater at various concentrations of sodium sulphate ions, where the measurement of the contact angles of each sample after treatment and the IFT between the oil model and the washing solutions were taken. The obtained results illustrated that bio-surfactants (rhamnolipids) with incremental concentrations of SO42− ions in sea water (up to three times higher than the original ion concentration) can lower the IFT, and assessed changing the rocks towards greater water-wettability. This study reveals that the alteration of SO42− ions had a greater impact on the wettability alteration, whereas rhamnolipids were better at reducing the IFT between the oil phase and the aqueous phase. This study also looked at temperatures of 50 °C and 70 °C, which demonstrated undesired influences on the wettability and IFT. Bio-surfactants at high temperature showed less interfacial activity, thus indicating that rhamnolipids are not active at high temperatures, while the addition of SO42− shows a continuous decrease in the contact angle and IFT measurements at high temperature.