The environmental biotic and abiotic factors form a complicated relationship with the host intestinal microbiota. In our study, we applied different levels of C/N ratio (10, 15, 20) and frequent addition times (once, twice, triple a day) in a factorial experimental design. GC/LC analysis of filtrated biofloc (BF) samples revealed the highest relative fold change for the untargeted bioactive molecules among different treatments, whereas the 16s rRNA analysis revealed the change in the shrimp gut microbiota composition. Based on the available literature on the relationship between the bioactive molecules and the available bacteria in this study, the next bioactive molecules were discussed. Proline was associated with Bacteroidota, Flavobacteriaceae, Gammaproteobacteria, and Flavobacteriales. Plumbagine was associated with Norcardiaceae. Phytosphingosin was associated with Bacteroidota. Phosphocholine compound was associated with Bacteroidota. The monobutyl ether, benzofuran, and piperidone were associated with Micobacteriaceae and Mycobacterium. Generally, C/N 15 and 20 once a day, and C/N 20 triple a day have showed a merit over other treatments in term of low pathogenic and unfavorable bacteria, and high commensal bacterial abundances. The revealed bioactive molecule composition showed the complicity of BF as a source for novel compounds as biosecurity agents in BF system. These molecules could be developed to feed additives upgrading the biosecurity level in aquaculture systems. Other bioactive molecules require future studies to reveal novel molecules in term of aquaculture biosecurity control.