Biofloc technology is commonly applied in intensive tilapia (Oreochromis niloticus) culture to maintain water quality, supply the fish with extra protein, and improve fish growth. However, the effect of dietary supplementation of processed biofloc on the gut prokaryotic (bacteria and archaea) community composition of tilapia is not well understood. In this study one recirculating aquaculture system was used to test how biofloc, including in-situ biofloc, dietary supplementation of ex-situ live or dead biofloc, influence fish gut prokaryotic community composition and growth performance in comparison to a biofloc-free control treatment. A core gut prokaryotic community was identified among all treatments by analyzing the temporal variations in gut prokaryotes. In-situ produced biofloc significantly increased the prokaryotic diversity in the gut by reducing the relative abundance of dominant Cetobacterium and increasing the relative abundance of potentially beneficial bacteria. The in-situ biofloc delivered a unique prokaryotic community in fish gut, while dietary supplementation of tilapias with 5% and 10% processed biofloc (live or dead) only changed the relative abundance of minor prokaryotic taxa outside the gut core microbiota. The modulatory effect of in-situ biofloc on tilapia gut microbiota was associated with the distinct microbial community in the biofloc water and undisturbed biofloc. The growth-promoting effect on tilapia was only detected in the in-situ biofloc treatment, while dietary supplementation of processed biofloc had no effect on fish growth performance as compared to the control treatment.