Increase in consumption of consumer items such as plasticizers have resulted in a sharp rise in the presence of xenobiotics like phthalic acid esters (PEs) in freshwater and marine environments due to contaminated runoff and improper release of effluents. The sublethal toxicity of Di-n-butyl phthalate (DBP) was investigated in juvenile Nile tilapia, Oreochromis niloticus, in an attempt to determine the biological effect of exposure to 1/2 and 1/3 median lethal concentration (96-h LC50) which, in our study was experimentally determined to be 11.8 mg/l. Following four days of exposure, indices of the oxidative potential [Malondialdehyde content (MDA)], antioxidant parameters [superoxide dismutase activity (SOD) and reduced glutathione level (GSH)] and DNA damage were evaluated by single-cell gel electrophoresis (Comet assay). Hepato-renal markers [alanine aminotransferase activity (ALT), creatinine and urea level] and cortisol levels were also quantified in serum. Additionally, histopathological investigations of liver, kidney and gill tissues were conducted. Comparative results between the 1/2 96-h LC50 group and the 1/3 96-h LC50 group clearly showed that there was a significant elevation in MDA levels and a marked increase in DNA damage in addition to inhibition of antioxidant barriers as represented by attenuation of SOD activity and GSH level in the group that was exposed to higher concentration of DBP (1/2 96-h LC50). The hepatorenal markers and cortisol levels were also observed to be elevated. Histopathological examination of the liver, kidney and gills showed pathological alterations that could be correlated with changes in the biochemical profile of the exposed fish. Additionally, anomalous clinical signs were noted. Based on these findings, we conclude from our study that exposure of juvenile O. niloticus to DBP has the potential to induce biochemical as well as tissue morphological alterations associated with oxidative injury and DNA damage.