Two freshwater macrophytes, water (Eichhornia crassipes) and coontail (Ceratophyllum demersum), were evaluated as carbon sources in biofloc system for Nile tilapia (Oreochromis niloticus), in comparison with molasses and a RAS with 10% water exchange daily (control). All-male tilapia (9.35 g) were stocked in 70-L fibreglass tanks at 10 fish tank −1 and fed on a commercial diet (30% cp) for 60 days. The average water temperature, pH and dissolved oxygen (DO) did not significantly differ (p > 0.05) among all treatments. Total ammonia-nitrogen (TAN) was significantly lower in the control group (0.11 mg L −1 ) than in biofloc treatments (0.15-0.17 mg L −1 ). Nitrite (NO 2 -N) concentrations were significantly lower, while nitrates (NO 3 -N) higher, in bioflocbased treatments (20.23-25.77 mg L −1 ) than in the control group (12.85 mg L −1 ). Biofloc volume (BFV) (28.17-33.50 ml L −1 ) and total suspended solids (TSS) (691-824 mg L −1 )were not significantly different among molasses and macrophytes treatments. The control group displayed lower growth rates and feed efficiency than macrophytesbased groups. Blood parameters, immunological responses, digestive enzymes activities, liver function enzymes and upregulation of IFNγ, TNFα and IL-8 genes were significantly better in molasses-and macrophytes-treated tanks than the control group. These results suggest that water hyacinth and coontail can be used as costless, eco-friendly carbon sources in biofloc systems.