© 2022 by the authors.This study was concerned with the green synthesis of gold nanoflowers (AuNFs) using the bioactive constituents of Rosmarinus officinalis (rosemary) and Helichrysum italicum (immortelle) extracts, as reducer and stabilizer agents along with the determination of their antibacterial and antibiofilm activity against E. coli, S. aureus, and S. epidermidis. The AuNFs were characterized using STEM, UV–Vis, DLS, ZETA, FESEM-EDX, and FTIR techniques. The antibacterial and antibiofilm activity of the AuNFs were evaluated by microdilution broth and microtiter plate (MTP) tests, respectively. STEM and DLS analysis confirmed the flower-like morphology of gold nanoparticle clusters of R. officinalis-AuNFs (R-AuNFs) and H. italicum-AuNFs (H-AuNFs) with a size of 20–130 nm and 15–90 nm, respectively. The MICs of R-AuNFs were found to be 40 µg/mL for E. coli and S. epidermidis and 160 µg/mL for S. aureus. The MICs of H-AuNFs against all bacterial strains were 20 µg/mL. All tested AuNFs exhibited a strong dose-dependent antibiofilm activity against the test strains, and H-AuNFs was more effective than R-AuNFs. The green synthesis of AuNFs from the rosemary and immortelle extracts can be applied as a potential agent to overcome the growth of biofilm-producing microorganisms in food industries.