While microbial fuel cells are being considered as a tool for energy saving in wastewater treatment facilities, such applications in oil refineries pose a challenge due to harder acclimation of microorganisms. In this research, the effect of heat pretreating mixed culture microorganisms (MCM), and cell cross section, on the performance of a novel cell design with two cross sections (single chamber microbial fuel cells, with circular: SCMFC_CC and rectangular: SCMFC_RC cross section) fed batched with refinery wastewater were investigated. First, using original and heat pretreated MCM, the performance of SCMFC_CC in terms of chemical oxygen demand (COD) removal and electricity production was investigated. Then, using only the heat pretreated MCM, the electricity production of SCMFC_RC was measured and compared with that of SCMFC_CC. Heat pretreatment of MCM improved maximum open circuit voltage (OCV) and maximum power density generated by 14% and 16%, respectively. However, heat pretreatment reduced COD removal by about 4%. The performance of SCMFC_CC in terms of maximum OCV and power density compared to SCMFC_RC was improved by 41% and 279%, respectively. Heat treatment of MCM increases the electricity generation of the cell, while reducing the performance of COD reduction due to decreasing the microorganism varieties in the MCM.