Di-n-butyl phthalate (DBP), one of the plasticizers, is considered a ubiquitous environmental contaminant due to its widespread application in personal-care products and serves as a raw material in many industries for the generation of many plastic products. Several scientific investigations have shown that DBP caused embryotoxicity and cognitive impairments. However, there is less understanding of the genotoxic potential of DBP in neuronal tissue when exposure happens continuously for several generations. The present study was undertaken to investigate the impact of DBP on the nucleic acids of neuronal tissue in one-month-old rats by performing a comet assay and biochemical analyses. By oral gavage, the parental generation (F) was administered DBP (500 mg/kg/day) during gestation (GD6-20) and lactation, and exposures were continued for three consecutive generations until the pups were grown to one-month-old. The oxidative stress assessments carried out in discrete brain regions isolated from one-month-old rats (F-F) following DBP exposure indicated significant inhibition in the levels of antioxidant enzymes (superoxide dismutase and catalase) while oxidant status (malondialdehyde) was elevated significantly. The extent of DNA damage using the comet assay, as measured by the olive moment, tail DNA percentage and tail length, was greater in DBP-treated rats compared with the control group, but RNA/DNA content decreased significantly. The results of this study suggested a strong link between oxidative stress and genetic integrity in the neuronal tissue of rats exposed to DBP generationally. To summarise, DBP exposure during pregnancy caused oxidative stress, which resulted in genetic instability in specific discrete brain regions of the third generation.