The interactions of typical nitrofuran antibiotics, nitrofurazone (NFZ) and nitrofurantoin (NFT), with bovine serum albumin (BSA) have been studied using fluorescence and FT-IR spectroscopy, circular dichroism (CD), atomic force microscopy (AFM) and molecular docking investigation. The results revealed that the fluorescence of BSA was quenched by nitrofurans with a static quenching mechanism. The conformation of BSA was changed and unfolded with the addition of nitrofurans because the alpha-helix and beta-sheet were varied to beta-turn and random structure. This study also indicated that the impact on the conformational changes of BSA caused by NFZ was more conspicuous than that of NFT. The molecular simulation studies were carried out to clearly describe the nature of the interaction between nitrofurans and BSA.