Sophorolipids (SLs) offer an “environmentally friendly” alternative to chemically produced surfactants currently used in formulations for crude oil extraction, processing, and reclamation. Studies herein describe how sophorolipid structure influences its interfacial properties for environmentally and industrially relevant oil–water systems where the oil phase is Arabian light crude oil, paraffin oil, decane, hexadecane, a 1:1 vol/vol mixture of o‐xylene and 1,2‐dimethylcyclohexane, or a mixture of paraffin oil, o‐xylene, and 1,2‐dimethylcyclohexane (synthetic crude oil). SL‐hexyl ester (SL‐HE) reduces the crude oil–water interfacial tension (IFT) by 57 and 91% at 0.001 and 0.5 mg/mL, respectively. Crude oil displacement tests reveal that SL‐ethyl ester (SL‐EE) and SL‐HE contract a crude oil slick on water to about 20% of its starting volume allowing for easier burning of spilled crude oil on marine surfaces. Water retention and emulsion phase (e.g., o/w vs. w/o) are determined by SL‐structure/concentration, oil concentration, and oil composition to understand their performance for crude oil transportation and clean‐up. For the first time, w/o emulsions were obtained using SLs and their formation occurred after homogenization when the oil phase consisted of a 1:1 mixture of o‐xylene and 1,2‐dimethylcyclohexane. Generally, the performance of SL‐esters in the above studies was superior to that using Triton X‐100, a comparison nonionic surfactant. Hence, SL‐esters offer a valuable platform for tuning interfacial properties to optimize surfactant performance.