The last step in the biosynthesis of flavin adenine dinucleotide (FAD) is considered a target for the design of antimicrobial drugs because it is carried out by two non-homologous proteins in eukaryotic and prokaryotic organisms. Monofunctional FMN: adenylyltransferases (FMNAT) in Eukarya and FMNAT modules of bifunctional FAD synthases (FADS) in Prokarya belong to different structural families with dissimilar chemistry and binding modes for the substrates. In this study, we analyzed the relevance of the hydrophobic environment of the flavin isoalloxazine in the FMNAT active site of Corynebacterium ammoniagenes FADS (CaFADS) through the mutational analysis of its F62, Y106, and F128 residues. They form the isoalloxazine binding cavity and are highly conserved in the prokaryotic FADS family. The spectroscopic, steady-state kinetics and thermodynamic data presented indicate that distortion of aromaticity at the FMNAT isoalloxazine binding cavity prevents FMN and FAD from correct accommodation in their binding cavity and, as a consequence, decreases the efficiency of the FMNAT activity. Therefore, the side-chains of F62, Y106 and F128 are relevant in the formation of the catalytic competent complex during FMNAT catalysis in CaFADS. The introduced mutations also modulate the activity occurring at the riboflavin kinase (RFK) module of CaFADS, further evidencing the formation of quaternary assemblies during catalysis.