Abstract Respiratory tract infections (RTIs) are a significant global health concern, further complicated by the rise of antibiotic resistance. In this study, we investigated a novel therapeutic approach by preparing and evaluating biocompatible herbal nanoparticles synthesized from Maerua Oblongifolia extract, a plant known for its potent antimicrobial and anti-inflammatory properties. The nanoparticles were synthesized using an eco-friendly green synthesis method, with Maerua Oblongifolia serving as a natural reducing and stabilizing agent. The synthesized nanoparticles were characterized using several techniques. Dynamic Light Scattering (DLS) revealed an average particle size of 80–150 nm, an ideal size for enhanced cellular uptake and bioavailability. Zeta potential analysis showed a value of -35 mV, indicating good colloidal stability. Scanning Electron Microscopy (SEM) images revealed spherical, well-dispersed nanoparticles with a smooth surface, while Transmission Electron Microscopy (TEM) confirmed the spherical morphology and uniform distribution of the bioactive compounds within the nanoparticles. X-ray Diffraction (XRD) patterns confirmed the semi-crystalline nature of the nanoparticles, with peaks corresponding to the phytochemicals from Maerua Oblongifolia. Fourier-Transform Infrared Spectroscopy (FTIR) detected key functional groups, such as hydroxyl and carbonyl, contributing to nanoparticle stabilization and bioactivity. Thermogravimetric Analysis (TGA) showed the nanoparticles were thermally stable up to 250°C, making them suitable for pharmaceutical applications. In vitro biocompatibility tests using the MTT assay on human lung epithelial cells (A549) demonstrated minimal cytotoxicity, indicating high biocompatibility even at higher concentrations.