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Bacterial pathogens cause pain and death, add significantly to the expense of healthcare globally, and pose a serious concern in many aspects of daily life. Additionally, they raise significant issues in other industries, including pharmaceuticals, clothing, and food packaging. Due to their unique properties, a great deal of attention has been given to biogenic metal nanoparticles, nanocomposites, and their applications against pathogenic bacteria. This study is focused on biogenic silver and copper nanoparticles and their composites (UL/Ag2 O-NPS, Ul/CuO-NPs, and Ul/Ag/Cu-NCMs) produced by the marine green alga Ulva lactuca. The characterization of biogenic nanoparticles UL/Ag2 O-NPS and Ul/CuO-NPs and their composites Ul/Ag/Cu-NCMs has been accomplished by FT-IR, SEM, TEM, EDS, XRD, and the zeta potential. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) experiments were conducted to prove antibacterial activity against both Gram-positive and Gram-negative bacteria and anti-biofilm. The FTIR spectroscopy results indicate the exiting band at 1633 cm−1, which represents N–H stretching in nanocomposites, with a small shift in both copper and silver nanoparticles, which is responsible for the bio-reduction of nanoparticles. The TEM image reveals that the Ul/Ag/Cu-NCMs were hexagonal, and the size distribution ranged from 10 to 35 nm. Meanwhile, Ul/CuO-NPs are rod-shaped, whereas UL/Ag2 O-NPS are spherical. The EDX analysis shows that Cu metal was present in a high weight percentage over Ag in the case of bio-Ag/Cu-NCMs. The X-ray diffraction denotes that Ul/Ag/Cu-NCMs, UL/CuO-NPs, and UL/Ag2 O-NPS were crystalline. The results predicted by the zeta potential demonstrate that Ul/Ag/Cu-NCMs were more stable than Ul/CuO-NPs. The antibacterial activity of UL/Ag2 O-NPS, Ul/Ag/Cu-NCMs, and UL/CuO-NPs was studied against eleven Gram-negative and Gram-positive multidrug-resistant bacterial species. The maximum inhibition zones were obtained with UL/Ag2 O-NPS, followed by Ul/Ag/Cu-NCMs and Ul/CuO-NPs in all the tested bacteria. The maximum anti-biofilm percentage formed by E. coli KY856933 was obtained with UL/Ag2 O-NPS. These findings suggest that the synthesized nanoparticles might be a great alternative for use as an antibacterial agent against different multidrug-resistant bacterial strains. [ABSTRACT FROM AUTHOR]