This work aimed to apply ultraviolet radiation as an alternative treatment to inhibit microbial growth in diesel fuel. Samples of fuel were placed in a reactor equipped with ultraviolet C (UV-C) radiation lamps (40 W, 253 nm) and a fuel recirculation system. Assays were performed using different UV-C exposure times (0-360 min), volume (40 and 60 L), and with / without a circulation system. The microorganisms present in diesel were quantified using the drop-plate technique and spectrophotometric detection (600 nm), and were identified by genetic sequencing. Parameters such as color, specific mass, and kinematic viscosity were evaluated to verify whether UV radiation promotes changes in phyco-chemical fuel properties. The assay with 360 min of radiation exposure, with a circulation system, caused the microorganism concentration to decrease from 1.50 × 10 10 to 3.33 × 10 2 CFU mL −1 , but otherwise did not lead to any significant changes in fuel physicochemical properties. Microorganisms of the genera Staphylococcus sp., Enterococcus sp., Clostridium sp., Enterobacter sp. and Bacillus sp. were identified. It could be concluded that the use of UV radiation as a microbiological control method resulted in a reduction of around 99% in the concentration of the microorganisms.