In Biofloc Technology (BFT), the disposal of solid waste represents a sustainability gap within. The presence of polyphenols in biofloc extracts has been reported, and these molecules have been widely explored for the synthesis of silver nanoparticles (AgNPs) in green nanotechnology. Polyphenols are employed to reduce silver ions and stabilize AgNPs. Compared to conventional methods (chemical synthesis), green synthesis makes the production of AgNPs sustainable and environmentally friendly by offering lower costs and avoiding the generation of hazardous waste that could impact the environment. This study proposes the extraction of polyphenols from bioflocs and their use as reducing and capping agents in the green synthesis of silver nanoparticles as a way to add value to the waste generated in BFT. Response Surface Methodology was used to obtain better extraction conditions by investigating the influence of four reaction variables (temperature, time, ethanol concentration, and solvent/solute ratios). The obtained extract was used to synthesize silver nanoparticles (BFAgNPs), which were used to determine the minimum inhibitory activity (MIC) and bactericidal activity (MBC) against Acinetobacter baumannii and Pseudomonas aeruginosa, as well as their anti-biofilm activity (inhibition and destruction) against A. baumannii. The highest polyphenol content was 1,918 μg gallic acid equivalents/g and the antioxidant activity was 1,278 μM Trolox equivalents/g for DPPH and 12,513 μM Trolox equivalents/g for ABTS. BFAgNPs exhibited an average diameter of 25.3 nm, as observed by transmission electron microscopy. Dynamic light scattering indicated a particle diameter of 123.2 nm, and polydispersity index (PDI) of 0.15. The zeta potential was measured at −22.5 mV. Fourier-transform infrared spectroscopy revealed distinct bands at 3616, 2924, 1682, 1532, 1234, and 1042 cm-1. BFAgNPs demonstrated MIC (38.8 μg/mL and 19.4 μg/mL for A. baumannii and P.aeruginosa), and MBC (77.5 μg/mL and 38.8 μg/mL for A. baumannii and P.aeruginosa) and antibiofilm activity, (47.3% inhibition and 27.8% destruction, respectively). It was concluded that the biofloc residue constitutes a source of polyphenols useful for the synthesis of AgNP with antibacterial capacity, thus representing a potential alternative for the waste valorization of BFT effluent. Display omitted •Biofloc residues generated in the BFT system are sources of polyphenols.•Polyphenols extracts were used to syntethize silver nanoparticles (AgNP).•AgNP exhibit inhibitory activity against Acinetobacter baumannii and Pseudomonas aeruginosa.•AgNP presented anti-biofilm activity against A. baumannii.•Biofloc residues show potential for the green synthesis of AgNP.