Introduction: Cigarette butts (CBs) are one of the most commonly found types of litter contaminating the aquatic environment. However, the environmental risks posed by CBs need further investigation. Therefore, it is necessary to evaluate the toxicity of the chemical contents of cigarette butts on aquatic organisms. Therefore, the aim of this study was survey the toxicity effects of chemical compositions of cigarette butts leachate on Periophthalmus waltoni fish. At first, the stock leachates and different dilutions of smoked cigarette butts with tobacco (SCB), smoked cigarette butts without tobacco (SF) and unsmoked cigarette butt (USF) leachates were prepared. Then, the lethal dose (LC50) values of the leachates of different forms of CBs were obtained by exposing the organisms to the prepared leachates. Fish were exposed to one-third of the predetermined 96h LC50 values of different types of leachates of CBs (SCBs, SFs, and USFs) in three acute (1 day), sub-acute (28 days), and sub-chronic (42 days) phases. After the determined times, the desired parameters (hematological parameters, blood serum enzymes, liver, gill, muscle and heart histology) were examined in the organisms. ICP-MS (Agilent, USA) and GC/MS were used to investigate the chemical composition of leachates, including heavy metals and PAHs, respectively. The mean concentration levels of PAHs in SCBs, SFs, and USFs leachates ranged from