The present study deals with the synthesis of selenium nanoparticles (SeNPs) using Nilgirianthus ciliatus leaf extracts, characterized by UV–Vis spectrophotometer, XRD, FTIR, FE-SEM, HR-TEM, DLS, and zeta potential analysis. The antimicrobial activity against Staphylococcus aureus (MTCC96), Escherichia coli (MTCC443), and Salmonella typhi (MTCC98) showed the remarkable inhibitory effect at 25 µl/mL concentration level. Furthermore, the characterized SeNPs showed a great insecticidal activity against Aedes aegypti in the early larval stages with the median Lethal Concentration (LC₅₀) of 0.92 mg/L. Histopathological observations of the SeNPs treated midgut and caeca regions of Ae. aegypti 4th instar larvae showed damaged epithelial layer and fragmented peritrophic membrane. In order to provide a mechanistic approach for further studies, molecular docking studies using Auto Dock Vina were performed with compounds of N. ciliatus within the active site of AeSCP2. Overall, the N. ciliates leaf-mediated biogenic SeNPs was promisingly evidenced to have potential larvicidal and food pathogenic bactericidal activity in an eco-friendly approach.