Mixture of fatty acids obtained from coconut, palm kernel, palm, jatropha, karanja, Sterculia foetida, and high oleic sunflower oils were used for the preparation of sodium N-acyl isoleucines (NaNAIle) via Schotten–Baumann reaction except castor oil which was prepared in thermal condensation in good yields. The products were characterized by chromatographic and spectral techniques. Surface active properties such as surface tension, wetting, foaming characteristics, emulsion stability, calcium tolerance, and micellization properties were evaluated for all the synthesized products. The results showed that all the products exhibited superior surface active properties like critical micelle concentration (CMC) and emulsion stability when compared to the commercial surfactant, sodium lauryl sulphate (SLS). The NaNAIle also exhibited promising cytotoxicity against human cancer cell lines except 3 and 5. 5, 6, and 8 showed antimicrobial activity against Gram-positive and Gram-negative bacteria. 1, 2, 7, and 8 exhibited good antioxidant properties.