For the first time, an aqueous extract of Melilotus officinalis was used to synthesize bimetallic silver selenide chalcogenide nanostructures (Ag2Se-NCs). The formation of NCs was confirmed and characterized by UV–visible and FTIR spectroscopy, SEM and TEM imaging, XRD and EDX crystallography, zeta potential (ZP) and size distribution (DLS). The bioactivities of biosynthesized Ag2Se-NCs, such as antibacterial, antibiofilm, antioxidant and cytotoxicity potentials, were then examined. Bio-based Ag2Se-NCs were successfully synthesized with mostly spherical shape in the size range of 20–40 nm. Additionally, the MIC and MBC values of Ag2Se-NCs against β-lactam-resistant Pseudomonas aeruginosa (ATCC 27853) were 3.12 and 50 µg/ml, respectively. The DPPH scavenging potential of Ag2Se-NCs in terms of IC50 was estimated to be 58.52. Green-synthesized Ag2Se-NCs have been shown to have promising benefits and could be used for biomedical applications. Although the findings indicate promising bioactivity of Ag2Se-NCs synthesized by M. officinalis extract (MO), more studies are required to clarify the comprehensive mechanistic biological activities.