The reduction of sulfur content in diesel is a worldwide trend that seeks to minimize the environmental impact arising from the combustion of this fuel in large cities. However, there is no reliable information on the effect of such a reduction on the formation of mainly aerobic microbial biomass in vehicle and storage tanks. The objective of this study was to assess the microbial community structure using next generation sequencing technique, and to evaluate the microbial biomass in pure diesel containing various sulfur concentrations. Storage conditions were simulated in glass flasks containing mineral medium and pure diesel, with varying sulfur content: UHSD (<= 1800 ppm sulfur); HSD (<= 500 ppm sulfur); LSD (<= 50 ppm sulfur) and ULSD (<= 10 ppm sulfur). Were assessed two microbial contamination levels in the diesel: low contamination (similar to 10(3) CFU L-1) and medium contamination (similar to 10(5) bacterial cells and fungal spores mL(-1)). The flasks were incubated at 30 degrees C and monitored every 10 days over 40 days. Microbial biomass formation was observed in all microcosms, but the highest biomass level at all times was in UHSD with medium contamination (p < 0.05). Results from biomass production and proton nuclear magnetic resonance showed that the ULSD, was not prone to microbial contamination probably because has highly branched carbon chain. Overall, 36 different phyla were identified by sequencing of 16S rDNA and four by 18S rDNA, with Firmicutes (similar to 49%), Bacteroidetes (similar to 25%), Actinobacteria (similar to 17%) and Ascomycota (>99%) being the most abundant. Results suggested that bacterial diversity is increased when low sulfur concentrations are present. Our results reveal a complex microbial diversity from 16S rDNA and confirm the existence of the major fungal Phyla (Ascomycota, Basidiomycota, Chytridiomycota and Glomeromycota) with a large fraction of microorganisms as yet undescribed, reinforcing the importance of understanding the fuel environment in establishing control methods of microbial growth in storage tanks and in bioremediation programs for environments contaminated by fuel. (C) 2016 Elsevier Ltd. All rights reserved.