The nontargeted action of fungicides affects the structure of protein, which leads to several serious diseases such as nausea, cancer, fetus malformations, movement dysfunction, and behavioral changes in human and animals. Hence, understanding of the structural change in protein induced by fungicides is of utmost importance to decode its mode of nontargeted action. In this study, we have investigated the structural change of myoglobin by an important fungicide, namely, dodine (n-dodecylguanidinium acetate), as well as its analogues n-hexylguanidinium acetate (HGA) and guanidinium chloride (GdmCl) using spectroscopic and thermodynamic methods. The amount of dodine and HGA required for the unfolding of myoglobin is significantly less than GdmCl. GdmCl, dodine, and HGA unfold the myoglobin by decreasing the content of the helical and tertiary structures. However, the decrease in the content of tertiary structure is significantly higher than that of the secondary structure for dodine and HGA, in contrast to GdmCl, where the decrease in secondary and tertiary contents of protein is not biased. Thermodynamic and spectroscopic data depict that the unfolding of the dodine and HGA is driven by the hydrophobic interaction, whereas the hydrogen bonding of GdmCl with the amino acids of protein plays a key role in the unfolding. The long alkyl chain of dodine and HGA get accommodated at the surface of the helices of myoglobin, inducing strong hydrophobic interaction, which causes its unfolding. This study depicts that dodine unfolds protein by the chaotropic effect in which its hydrocarbon chain destabilizes the protein by the hydrophobic effect, unlike in an earlier study, where dodine was claimed to be a kosmo-chaotropic agent as its hydrocarbon group stabilizes the protein.