To understand the microbiome composition and interplay among bacterial communities in different compartments of a coupled freshwater aquaponics system growing flathead grey mullet (Mugil cephalus) and lettuces (Lactuca sativa), 16S rRNA gene amplicon sequencing of the V3–V4 region was analysed from each compartment (fish intestine, water from the sedimentation tank, bioballs from the biological filter, water and biofilm from the hydroponic unit, and lettuce roots). The bacterial communities of each sample group showed a stable diversity during all the trial, except for the fish gut microbiota, which displayed lower alpha diversity values. Regarding beta diversity, the structure of bacterial communities belonging to the biofilm adhering to the hydroponic tank walls, bioballs, and lettuce roots resembled each other (weighted and unweighted UniFrac distances), while bacteria from water samples also clustered together. However, both of the above-mentioned bacterial communities did not resemble those of fish gut. We found a low or almost null number of shared Amplicon Sequence Variants (ASVs) among sampled groups which indicated that each compartment worked as an independent microbiome. Regarding fish health and food safety, the microbiome profile did not reveal neither fish pathogens nor bacterial species potentially pathogenic for food health, highlighting the safety of this sustainable food production system.