The aim of this study was to evaluate the safety, antioxidant potential, and antimicrobial properties of the aqueous extract (AE) of Juglans regia L. bark collected in the Tizi-Ouzou region in Algeria. The richness of AE in polyphenols was assessed by RP-HPLC. The biological activities of AE were expressed through their antioxidant, antibacterial, antibiofilm, and antihemolytic potentials. 18 phytochemical molecules (12 flavonoid molecules and 6 phenolic acid molecules) were identified by RP-HPLC. The antioxidant activity expressed by TAC was 119.08± meq AA/g of DW, while the results of DPPH and FRAP expressed by IC50 and EC50, were 57±1.02 µg/ml and 65±0.67 µg/ml, respectively. AE showed antibacterial activities against all tested strains: E. coli ATCC 25922, B. cereus ATCC27853, S. aureus ATCC25923, E. faecalis WDCM0009, and P. aeruginosa ATCC 27853. The biofilm of S. aureus ATCC25923 was inhibited by 93% at a biofilm inhibitory concentration of 56.25 mg/ml. AE was non-hemolytic and protected the erythrocyte membrane against hemolysis caused by saponin. The results of this study demonstrate that walnut bark can be integrated into pharmaceutical formulations, especially those intended for oral hygiene and cosmetic products.