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Introduction: Nowadays tramadol is the most common drug of abuse. Egyptian surveys found a gradual increase in the use of tramadol among Egyptians. It has been associated with a wide range of drug abuse such as benzodiazepines. Aim of the work: This study aimed to evaluate the effects of dependence of tramadol, diazepam, and their combination on biochemical, histopathological and immunohistochemical changes of brain of adult albino rats.Material and Methods: Forty adult male albino rats were divided into four equal groups as follows: Group I (control) received1ml normal saline (0.9%NaCl) once orally for one month. Group II (tramadol dependent) received increasing therapeutic doses of tramadol orally for one month, Group III (diazepam dependent) received increasing therapeutic doses of diazepam orally for one month, Group IV (tramadol and diazepam dependent) received increasing therapeutic doses of tramadol and diazepam orally for one month. Blood samples were collected from all groups for evaluation of serum cortisol level. Brain was excised for biochemical, histopathological and immunohistolochemical studies. Results: Compared to the control group, serum cortisol level was significantly decreased in tramadol dependent and combined tramadol and diazepam dependent groups. In all experimental dependent groups, brain cholinesterase level was not changed and the brain showed histopathological and immunohistochemical changes Conclusion and recommendation: Tramadol or diazepam dependence for long time affects the brain cells and the combination of both of them leads to more neurotoxic effect. Therefore it is recommended that tramadol or diazepam should be taken only with the prescription of doctor and self medication of these drugs may be hazardous.