The present study was conducted to estimate the effect of cadmium chloride as a heavy metal and commercial glyphosate (Roundup®) as a herbicide on DNA damage as biochemical and molecular biomarkers in the Nile tilapia, Oreochromis niloticus through three exposure periods with different concentrations of LC₅₀. The 96h-LC₅₀ were determined for CdCl₂ (132mg/l), glyphosate (9.63mg/l), CdCl₂ in mixture (41.30mg/l) and glyphosate in mixture (2.75mg/l), respectively. The fish exposed to these concentrations separately and mixed for 4 days as well as two sublethal concentrations (1/4 and 1/10 LC₅₀) for 8 days and 45 days, respectively. Gills and liver cells of the exposed samples were taken after 4, 8 and 45 days to examine the DNA damage using comet assay. The highest DNA damage was observed on gills after 4 days in treatment of mixture due to presence of synergistic effect between CdCl₂ and glyphosate , after 8 days in treatment of glyphosate and after 45 days in treatment of cadmium. The highest DNA damage was observed on liver after 4days and 8days in treatment of glyphosate but after 45days in treatments of cadmium and mixture. DNA damage depends on concentration of pollutants and exposure periods.