Mosquitoes are the primary vector for transmitting endemic diseases such as yellow fever, Chikungunya, dengue and dengue hemorrhagic fever, malaria, Japanese encephalitis, and lymphatic filariasis in humans and animals. The present study was carried out by the myco-synthesis of silver nanoparticles (AgNPs) using entomopathogenic fungi Beauveria bassiana and its mosquitocidal properties against different larval instars of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. The synthesized AgNPs were characterized by UV-visible spectroscopy, X-ray diffraction analysis (XRD), Fourier transform infrared spectroscopy (FTIR), Particle Size Analyzer (PSA), Transmission Electron Microscope (TEM), and Energy Dispersive X-ray spectroscopy (EDX), respectively. The surface plasmon resonance band was observed at 430 ± 1.5 nm in the UV-spectrum. The XRD spectrum of crystallic AgNPs shows four strong intense peaks at 2θ values of 38.12°, 54.14°, 64.18°, and 77.48° assigned to 111, 200, 220, and 311, respectively. The complex nature of AgNPs was confirmed by an FTIR spectrum with peaks observed at 3205.77, 1653.20, 1383.50, 1071.12, 825.58, 668.85, and 565.17 cm⁻¹ respectively. The average size of synthesized AgNPs was confirmed by PSA (128 nm). TEM images confirmed the AgNPs size about 20.44 to 34.16 nm (spherical shape) and EDX spectral peak was observed between 1.741 to 5.430 keV. The larvicidal efficacy of different concentrations (50, 100, 150, 200, 250, and 300 μg mL⁻¹) of mycosynthesized AgNPs was tested against first, second, third, and fourth larval instars of A. aegypti (LC₅₀ = 34.655, 20.522, 22.621, and 22.536; LC₉₀ = 170.207, 144.763, 120.934, and 198.394 μg mL⁻¹), A. stephensi (LC₅₀ = 45.822, 16.777, 22.873, and 30.787; LC₉₀ = 298.812, 111.038, 142.154, and 193.092 μg mL⁻¹), and C. quinquefasciatus (LC₅₀ = 49.36, 26.946, 13.199, and 1.478; LC₉₀ = 161.554, 134.590, 104.639, and 58.923 μg mL⁻¹) and 100% mortality effect was observed with the AgNPs. The antibacterial property of synthesized nanoparticles was observed by the well diffusion method with multi-drug resistant bacteria such as Escherichia coli and Staphylococcus aureus. The fungal mediated silver nanoparticles are comparatively rapid and less expensive and have broad application to antibacterial therapy in modern medicine. The cytotoxic effects of silver nanoparticles showed IC₅₀ values of 28.8 ± 2.5 μg mL⁻¹ against normal HeLa (cervical) cells, with a 24 h incubation period. The outcome of the study suggests that the B. bassiana synthesized silver nanoparticles would be more appropriate for environmentally safer bio insecticidal agents for controlling A. stephensi and C. quinquefasciatus mosquitoes.