Chloramphenicol was discovered from Streptomyces venezuelae and synthetically produced in 1948. This investigation aimed on the effect of long-term oral administration of chloramphenicol on hepato-renal and oxidative stress biomarkers in Sprague dawley rats. After each of the ten rats in the experimental group consumed 250 mg of chloramphenicol dissolved in 15 milliliters of distilled water on a daily basis for 30 days, five milliliters of blood specimen were taken from each of them and dispensed into lithium heparin anticoagulated bottle respectively. The remaining ten rats, which served as controls, were not administered with chloramphenicol or any other antibiotics. The rats in the experimental group demonstrated mean values of alanine aminotransferase (21.16 ± 2.81) U/L, aspartate aminotransferase (20.27 ± 2.60) U/L,, alkaline phosphatase (37.10 ± 2.92) U/L, creatinine (32.17 ± 0.88) µmol/L, urea (6.74 ± 0.24) mmol/L, glutathione peroxidase (4.78 ± 0.61) µmol/L and malondialdehyde (4.71 ± 0.57) mmol/L as being statistically significant when compared to that of the control group alanine aminotransferase (4.02 ± 0.83) U/L, aspartate aminotransferase (3.97 ± 0.64) U/L, alkaline phosphatase (12.80 ± 1.75) U/L, creatinine (32.12 ± 0.87) µmol/L, urea (6.71 ± 0.22) mmol/L, glutathione peroxidase (2.15 ± 0.10) µmol/L and malondialdehyde (2.02 ± 0.06) mmol/L respectively. In conclusion, taking 250 mg of chloramphenicol diluted in 15 milliliters of distilled water every day for 30 days may trigger hepato-oxidative stress disorders in Sprague dawley rats