Environmental and economic concerns accelerated biofuels research and industrial production. Many countries have been using diesel and biodiesel blends as fuels justifying research on biofilms formation and metals corrosion. Cylinders made of AISI-1020 carbon steel with an exposed area of 1587 mm2, water, and water associated with B3 fuel (diesel/biodiesel blend at 97 : 3 v/v) were used.The formation of biofilms was detected, and biocorrosion was detected on AISI-1020. The results showed a variation in sessile microflora during the experiments. In the biofilms, a significant concentration of aerobic, anaerobic, IOB, Pseudomonas aeruginosa, and sulfate-reducing bacteria was observed. The corrosion rates varied between 0.45±0.01 and 0.12±0.01 mm/year, depending on the experimental conditions. The main corrosion products identified were various forms of FeOOH, magnetite, and all forms of FexSy. In systems where there were high levels of sulfate reducing bacteria, corrosion pits were observed. In addition, the aliphatic hydrocarbons present in the fluid containing 10% B3 were totally degraded.