Effective biosynthesis of magnetite nanoparticles using current technology is challenging. We investigated the synthesis of nanoparticles by Acidithiobacillus ferrooxidans grown on ferrous iron, elemental sulphur, and mixtures of both substrates. A comparison of tests with different doping amounts of elemental sulphur in ferrous-containing medium showed that the addition of 0.25 and 0.5 M elemental sulphur to the medium resulted in an increased delay of microbial growth and ferrous iron oxidation. TEM suggested that the ferrous material was an essential energy source for the synthesis of nanoparticles in cells. TEM results indicated that the different ratios of ferrous and sulphur had no significant effect on the morphology of bacteria and the size of nanoparticles. High-resolution transmission electron microscopy (HRTEM), energy dispersive X-ray spectroscopy (EDX), and X-ray absorption near edge structure (XANES) showed that the nanoparticles were composed of magnetite. For the first time, HRTEM and XANES spectra in-situ characterization was conducted to investigate the nanoparticles that were synthesized by A. ferrooxidans. The findings from this study indicated that the different ratios of ferrous and sulphur had no significant effect on size and shape of nanoparticles synthesized by A. ferrooxidans.