Biofilm forming from a variety of microbial pathogens can pose a serious health hazard that is difficult to combat. Nanotechnology, however, represents a new approach to fighting and eradicating biofilm-forming microorganisms. In the present study, the sustainable synthesis and characterization of biocompatible silver nanoparticles (AgNPs) from leaf extracts of Semecarpus anacardium, Glochidion lanceolarium, and Bridelia retusa was explored. Continuous synthesis was observed in a UV-vis spectroscopic analysis and the participating phytoconstituents, flavonoids, phenolic compounds, phytosterols, and glycosides, were characterized by Attenuated total reflectance-Fourier transform infrared spectroscopy. The size and surface charge of the particles were also measured by dynamic light scattering spectroscopy. Scanning electron microscopy study was employed to examine the morphology of the nanoparticles. The spectroscopic and microscopic study confirmed the successful synthesis of AgNPs by plant extracts acting as strong reducing agents. The synthesized AgNPs were screened for antibacterial and anti-biofilm activity against human pathogens Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus. Results of the study demonstrate the potential of phyto-synthesized AgNPs to act as anti-biofilm agents and for other biomedical applications.