Anionic biosurfactant surfactin-mediated gold nanoparticles were synthesized for the first time in this study. Differing proton concentrations is believed to cause structural changes in the lipopeptide surfactin used to stabilize the gold nanoparticles in aqueous solution, the effects of which on the morphology of the nanoparticles were investigated. Synthesis of gold nanoparticles by borohydrate reduction was performed at three pH levels (pH 5, 7 and 9) and two different temperatures, and the nanoparticles were characterized by UV-visible spectroscopy, X-ray diffraction and transmission electron microscopy. The UV-vis spectra showed a blue shift with increasing pH from 5 to 9 (from 528 to 566 nm) at both 4 degrees C and room temperature. The nanoparticles synthesized at pH 7 and 9 remained stable for 2 months, while aggregates were observed at pH 5 within 24 h. TEM micrographs revealed that the mean particle size was about 13.11, 8.16 and 4.70 nm at pH 5, 9 and 7, respectively, at 4 degrees C. The nanoparticles formed at pH 7 were uniform in shape and size, and polydispersed and anisotropic at pH 5 and 9. The nanoparticles synthesized at room temperature were monodispersed and were more uniform as compared with those formed at 4 degrees C. This report describes the use of a renewable and environmentally green and biodegradable surfactant as a template and stabilizing agent in the synthesis of gold nanoparticles.