The present research aims to valorize biofloc waste by extracting polyphenols via alkaline hydrolysis, utilizing them as reducing and stabilizing agents in the optimization of green synthesis of silver nanoparticles (BIOAgNPs). For the alkaline extractions, potassium hydroxide (KOH) concentrations of 0 to 4 M were used in combination with ultrasound. Total polyphenol content and antioxidant activity were evaluated. BIOAgNPs synthesis was optimized using the response surface methodology and central composite design. The parameters time, temperature, AgNO₃ concentration, and the percentage of the biofloc extract (2 M KOH), with measurements taken for the area under the curve (AUC) (400–500 nm) and the mean hydrodynamic diameter (DLS), were evaluated. Antibacterial activity was determined for Gram-negative and Gram-positive bacteria. Higher polyphenol content and antioxidant activity were observed using 2 M KOH. The optimized model for DLS and the AUC was obtained in 4 h, at 40 °C, using 2.4 mM AgNO₃ and obtaining 2.5% of extract. Optimized BIOAgNPs had a diameter of 22.4 nm, hydrodynamic diameter of 106.5 nm, zeta potential of −28 mV, and polydispersity index of 0.26. BIOAgNPs demonstrated bactericidal activities. This study enhanced the valorization of aquaculture residues through improved polyphenol extraction techniques and developed an effective methodology for synthesizing silver nanoparticles with antimicrobial activity.