This study aimed to shed light on the effect of eszopiclone (ESZ) administration once daily for 30 consecutive days at night time on some neurochemical, behavioral and hematological criteria. A total of 27 male Wister albino rats were assigned to one of three drug treatment groups, vehicle, Eszopiclone (3mg/kg) and (6mg/kg). After 30 days of Eszopiclone administration, the neurochemical analysis revealed a significant reduction in serotonin and glutamate (306.44 ng/ml, 4.33 nmol /μl, respectively) in 6mg/kg treated animals, furthermore dopamine levels were significantly higher in rats treated with Eszopiclone (3mg/kg or 6mg/kg) in compare to control group. Reduced glutathione, superoxide dismutase and catalase levels revealed a significant decrease (0.15 μmol /gm tissue, 25.24 μmol /gm tissue, 1.93 Unit/gm tissue, respectively), while the malondialdehyde levels (15.79 nmol /gm tissue) demonstrated a significant increase in animals treated with 6 mg of ezopiclone. Behavioral assessment was carried out 3 times throughout the study (once/2 weeks) by video recording. It was recorded in 5 tests, including open field, the hole-board, inclined plain, grip and tail suspension tests. There were no changes between the rats in 3 mg/kg of ezopiclone and those in the control group. The rats in 6 mg/kg of ezopiclone showed less response in all behavioral observations, with significant decreases in inclined plain angle (23.35), exploratory time (17 second) and exploratory frequency (2 frequencies) in compare to other groups. The results support the concept that the administration of eszpiclone more than 3 mg/kg may lead to the behavioral changes. There were no serious adverse events regarding hematological indices. It is concluded that eszopiclone administration causes an imbalance between different neurotransmitters in the cerebrum. A marked decrease in antioxidant scavenging capacity with a behavioral alteration in 6mg/kg treated animals. The maximum safe dose of eszopiclone was 3mg/kg and more than this dose could lead to a deleterious reactions as evidenced in this study.