Microbial growth inside fuel storage tanks causes serious financial and operational problems. We assessed the relationship of two biocides with the diesel microbiome in five storage tanks. After 35 days of treatment, 16S rDNA amplicon sequencing was performed. Sequences were clustered into operational taxonomic units using QIIME and metagenome functional profile was inferred using PICRUSt. Proteobacteria was the predominant phylum in all samples. In general, Burkholderia, Stenotrophomonas, Ralstonia, and Acinetobacter were the most abundant genera. Five less frequent genera and four pathways were differently distributed among groups (p < 0.05). Using next generation sequencing, it was possible to detect uncultured, less frequent diesel associated taxa and specific pathways possibly leading to biocide resistance. Despite being a currently used strategy in the management of diesel tanks to avoid biodeterioration, the biocides tested were not effective in causing significant changes in the microbiota.