In this study, Escherichia coli DH5α biofilm was evaluated for its potential to control and minimize microbiologically influenced concrete deterioration (MICD) under mesophilic temperatures (37 °C). Escherichia coli DH5α biofilm was first grown on Portland cement mortar disks for 8 days. Mortar disks were then exposed to two different types of sulfur oxidizing bacteria (SOB) (Thiobacillus neapolitanus and Thiobacillus thiooxidans), which use sulfur compounds as substrate and oxidize them to sulfate and sulfuric acid. The effectiveness of the biofilm against MICD was evaluated by measuring pH, sulfate, calcium concentrations in the reactors and surface analysis of the mortar samples using X-ray diffraction and visual inspection. Overall, the results indicate that the E. coli DH5α biofilm showed good protection against MICD induced by SOB at 37 °C.