Biosynthetic mackinawite (FeS) has high removal efficiency for in situ remediation of heavy metal contamination. However, the effect of the iron (Fe) source on the efficiency of biosynthesized nano-FeS (nFeS) is less known. In this study, we examined the effect of Fe(II) produced i) directly from iron-reducing bacteria (IRB) Shewanella oneidensis MR-1 (HM group), and ii) indirectly from sulfate-reducing bacteria (SRB) Desulfosporosinus meridiei (DM group) on the biosynthesis of mackinawite, focusing on the biomineralization process and its capacity to remove hexavalent chromium (Cr(VI)). The HM group formed minerals around the cells, whereas the DM group formed minerals in the solution. Scanning electron microscopy, transmission electron microscopy, Xray diffraction, and X-ray photoelectron spectrometry analyses further showed that the Fe(II) produced directly (by MR-1, HM group) and indirectly (reduced by biogenic H2S, DM group) led to a different distribution of Fe(II) in FeS minerals. The removal capacities of the produced HM-nFeS and DM-nFeS at pH 3 were the same, with a difference of approximately 20% with an increase in pH from 5 to 11, further indicating that the formed FeS products were different. The MR-1 (HM group) directly reduced Fe(III) and secreted a large amount of organic matter, which may have inhibited the ability of the surface to adsorb HCrO- and CrO42-. These findings shed new light on the biosynthesis of mackinawite for bioremediation of heavy metals.