Identification of the core microbiome of real fuel tanks through bacterial and fungal biodiversity analyses (Illumina sequencing of ribosomal markers) has been performed. Results have been used to create a microbial strain collection (142 strains). From those, ten strains have been selected following two criteria: i) belonging to OTUs from the core microbiome and ii) belonging to genera defined in literature as problematic for fuel systems. After defining antagonisms between strains, a three-strain consortium comprehended by a fungal strain (genus Amorphotheca), and two bacterial strains (genera Pseudomonas and Escherichia) has been evaluated for its ability to form a synergistic biofilm on the surface of a representative material of aircraft fuel tanks. The multispecies biofilm growth was optimized and tested at lab-scale, simulating real environmental conditions, and monitored through plate count and Scanning Electron Microscope (SEM).