Antibiotic resistance has emerged as a critical global health challenge, complicating the treatment of infectious diseases. The bioactive compounds present in plants offer promising avenues for addressing antibiotic resistance, particularly when combined with advanced biotechnological approaches. This study aimed to develop plant extract-loaded electrospun nanofibers for preventing biofilm-associated infections, with a focus on the antimicrobial and antibiofilm properties of Astragalus lusitanicus subsp. orientalis. Phytochemical analysis of the plant extract was conducted using liquid chromatography-electrospray tandem mass spectrometry, identifying hesperidin (7864 µg/g) as the most abundant compound, followed by quercetin (2223 µg/g) and hyperoside (1804 µg/g). Electrospun nanofibers were successfully fabricated, incorporating the plant extract, and characterized using scanning electron microscopy, and Fourier-transform infrared spectroscopy. In vitro tests revealed the nanofibers’ favorable properties, including uniform morphology, flexibility, and ease of handling. Anti-microbial activity was evaluated via disc diffusion and broth microdilution assays, demonstrating significant effects against Klebsiella pneumoniae (24±1.25 mm zone of inhibition) and other pathogens at varying concentrations. Notably, Staphylococcus aureus ATCC 25923 and Bacillus subtilis NRS 744 were resistant. Antibiofilm assays showed that plant extract-loaded nanofibers and the plant extract inhibited biofilm formation by 96% and 82%, respectively, against biofilm-producing Staphylococcus aureus ATCC 25923, as confirmed by SEM. This study highlights the potential of Astragalus lusitanicus subsp. orientalis as a source of antimicrobial agents and demonstrates the synergistic benefits of integrating its extract into nanofibers. The findings provide a foundation for industrial applications in antimicrobial therapies and tissue engineering, emphasizing the high biocompatibility and controlled release properties of the nanofiber system. For the first time, the antibiofilm effects of this plant species are reported, underscoring its potential in accelerating the healing process and combating biofilm-associated infections.