Microbially-mediated nitrate-reducing Fe(II) oxidation (NRFO) is ubiquitous under neutral anaerobic iron-rich environments. However, it is still unknown whether NRFO can be mediated by dissimilatory iron reducing bacteria. In this study, Klebsiella pneumoniae L17, Shewanella oneidensis MR-1, and Shewanella putrefaciens strain CN32 were used as the model iron reducing strains to study the process of anaerobic NRFO. The results show that nitrate reduction and Fe(II) oxidation occurred simultaneously. The intermediate product nitrite during nitrate reduction is considered the dominant contributor to the overall Fe(II) oxidation. The presence of Fe(Ⅱ) inhibited microbial nitrate reduction, and the inhibitory effect of Fe(Ⅱ) was more significant with the higher Fe(Ⅱ) concentration. The competition between chemical reduction of nitrite by Fe(Ⅱ) and biological nitrite reduction resulted in the decrease of ammonium production. Iron oxides precipitated on the cell surface and subsequently prevented nitrate uptake and microbial nitrate reduction. The cell toxicity of Fe(Ⅱ) and secondary minerals inhibited biological nitrate reduction by L17 with low Fe(Ⅱ) concentration. Nitrate reduction by CN32 was inhibited by the secondary minerals during Fe(II) oxidation while the inhibition of nitrate reduction by MR-1 was attributed to cell toxicity of Fe(Ⅱ). Under high Fe(Ⅱ) concentration, microbial nitrate reduction was inhibited by cell encrustation. This study emphasizes iron-reducing bacteria mediated nitrate-reducing Fe(Ⅱ) oxidation in the anaerobic iron-rich environment, and expands the scope of microbial species with the ability of nitrate-reducing Fe(Ⅱ) oxidation. Therefore, the findings would be helpful for understanding the function and relative contribution of various microorganisms in the process of nitrate-reducing Fe(Ⅱ) oxidation in anaerobic environment.