In this study, the phytochemical profile and silver nanoparticle (AgNPs)-synthesizing ability of Pittosporum undulatum methanol extract were investigated. Furthermore, biological applications of the AgNPs, such as antibacterial effect (against Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilis, and Escherichia coli), mosquito larvicidal effect (against Anopheles stephensi, Culex quinquefasciatus, and Aedes aegypti), and cytotoxicity (against fibroblast cell line L929) were evaluated using in vitro experiments. The phytochemical analysis revealed that the methanol extract contained cardiac glycosides, terpenoids, saponins, alkaloids, flavonoids, glycosides, coumarins, phenolics, and tannins. Furthermore, standard characterization techniques such as UV–Vis spectrometry, SEM, TEM, FTIR, and XRD confirmed that the methanol extract of P. undulatum effectively synthesized the AgNPs. The synthesized AgNPs had a spherical shape and size of 20–200 nm. The bactericidal analysis revealed that the AgNPs have dose-dependent antibacterial activity. The MTT assay showed that the AgNPs were bio-compatible up to a dosage of 250 μg mL−1 in the normal fibroblast cell line L929. Furthermore, the LC50 values for AgNPs against larvae of An. stephensi, Cx. quinquefasciatus, and Ae. aegypti were 0.4, 4.7, and 1.2 ppm, respectively. Field trials demonstrated that the larvicidal effect was enhanced within 24–72 h, and the rate of reduction increased over time. Thus, our findings provide an ideal sustainable AgNP bio-pesticide to combat filarial, dengue, and malaria vectors.