Background Exposure to pesticides is associated with an increase in the incidence of cancer. We aimed to investigate the association of serum organochlorine pesticides (OCPs) and organophosphorus pesticides (OPs) levels and GSTM1/GSTT1 gene polymorphism with bladder cancer (BC). Methods This study was performed on 57 patients with BC and 30 controls (C). Acetylcholinesterase (AChE) activity, arylesterase activity of paraoxonase‐1 (ARE), total antioxidant capacity (TAC), and malondialdehyde (MDA) levels were determined in serums of all participants. Genomic DNA was extracted using the salting out method and GSTM1/GSTT1 gene polymorphisms were examined by multiplex polymerase chain reaction assay. Measurement of OCPs (α‐hexachlorocyclohexane [α‐HCH], β‐HCH, γ‐HCH, 2,4‐dichlorodiphenyltrichloroethane [2,4‐DDT], 4,4‐DDT, 2,4‐ dichlorodiphenyldichloroethylene [2,4‐DDE], and 4,4‐DDE) in serum was carried out using an FID‐equipped gas‐chromatography system. Results AChE activity was significantly lower, ARE activity and TAC were declined but it was not statistically significant, however, α‐HCH, γ‐HCH, 4,4‐DDE, 2,4‐DDT, and 4,4‐DDT pesticides, and MDA were significantly higher in BC patients compared with the control subjects. Also, a positive correlation was found between the number of smoked cigarettes and the years of smoking with BC development. There was no association between GSTM1/GSTT1 gene polymorphisms and OCPs in BC patients. Conclusion Due to the higher levels of some OCPs in the BC patients, along with the reduction in AChE activity and increased MDA levels, it may be concluded that OCPs and OPs play an important role in the induction of BC in southeastern Iran.