Background: Silver (Ag) and selenium (Se) nanoparticles are known for their unique antimicrobial properties. This study aims to synthesize Silver-Selenium (Ag-Se) nanoparticles using endophytic fungi and evaluate their antibacterial and anti-biofilm activities against clinically relevant bacterial strains. Methods: Endophytic fungi were isolated from various plant samples, and Ag-Se nanoparticles were synthesized through a green synthesis method involving the reduction of silver nitrate (AgNO₃) and sodium selenite (Na₂SeO₃) using fungal extracts. The synthesized nanoparticles were characterized using techniques such as SEM, TEM, DLS, FTIR, and XRD. Antibacterial activity was assessed via the agar well diffusion method and Minimum Inhibitory Concentration (MIC) determination. Anti-biofilm activity was evaluated using a microtiter plate assay to quantify biofilm inhibition. Results: The Ag-Se nanoparticles were characterized as spherical with an average size of 20-50 nm and exhibited a zeta potential of -27.3 mV, indicating good stability. The nanoparticles demonstrated significant antibacterial activity, with zones of inhibition of 16.1 mm against Escherichia coli, 21.0 mm against Staphylococcus aureus, and 14.8 mm against Pseudomonas aeruginosa. MIC values for Ag-Se nanoparticles were 25 µg/mL for E. coli, 30 µg/mL for S. aureus, and 40 µg/mL for P. aeruginosa, significantly lower than those for individual Ag (50, 60, and 70 µg/mL, respectively) and Se nanoparticles. Biofilm inhibition percentages were 65% for E. coli, 70% for S. aureus, and 58% for P. aeruginosa, with statistical significance confirmed by ANOVA and Tukey's post hoc test (p < 0.01). Conclusion: The study demonstrates that Ag-Se nanoparticles synthesized from endophytic fungi exhibit superior antibacterial and anti-biofilm properties compared to their counterparts. These findings suggest the potential of Ag-Se nanoparticles as effective agents in combating bacterial infections and biofilm-related challenges, paving the way for their application in medical and industrial settings.