The non-targeted action of fungicides leads to DNA damage of organisms, which causes several serious diseases such as cancer, behavioral change, and nausea. The mechanistic aspects of DNA damage by fungicides are not much known, irrespective of its importance. Spectroscopic and molecular dynamics simulation techniques have been employed to bring out the key aspects of the mechanism of DNA damage by an important fungicide, namely, n-dodecylguanidine acetate (dodine). The hydrocarbon and guanidinium groups of dodine interact in the adenine (A)-thymine (T) region of the minor groove of DNA via electrostatic and hydrophobic interactions. The accommodation of dodine in the minor groove of DNA interrupts the cone of hydration of DNA by removing water from its surface. The interaction of dodine in the minor groove of DNA perturbs inclination, twist, roll, and slide of base pairs in the A-T region, which broadens the minor groove as well as elongates the first strand of DNA, causing the loss of its helicity and base stacking. The detailed understanding of dodine-induced DNA damage may guide the development of fungicide with minimal non-targeted genotoxic effect.