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In this work, we reported the synthesis of honey bee (Apis mellifera) venom-derived nanoparticles via a hydrothermal method. This method not only ensures the preservation of the bee venom's bioactive components but also enhances their potential stability, thus broadening the scope for their applications in the biomedicinal field. The synthesis method started with the homogenization suspension of bee venom, followed by its hydrothermal process to synthesize bee venom nanoparticles (BVNPs). The successful synthesis of BVNPs was characterized using various characteristic techniques such as Ultraviolet-visible (UV-Vis) spectroscopy, Fourier Transforms Infrared (FTIR) Spectroscopy, Zeta Potential (ZP), Liquid Chromatography-Mass Spectrometry (LCMS), and Transmission Electron Microscopy (TEM). The synthesis of BVNPs through biosynthesis is shown by the visible violet-brown color development at 347 nm by UV-Vis spectroscopy. FTIR analysis revealed the presence of several functional groups in the BVNPs, including alcohols (-OH), phenols (C 6 H 5 -), carboxylic acids (-COOH), amines (-NH 2 , -NH-), aldehydes (-CHO), ketones (-CO-), nitriles (-CN), amides (-CO-N-), imines (-CNH-), esters (-COO-), and polysaccharides. These functional groups, as confirmed by their specific stretching and bending vibrational modes, contribute to the diverse biological activities of BVNPs, including cytotoxicity against MCF-7 breast cancer cells. The ZP of the BVNPs indicated good colloidal stability at - 45 mV. LCMS analysis confirmed the presence of major bioactive molecules, including melittin & apamin and TEM analysis shows the BVNPs exhibited a quasi-spherical shape with good dispersion, the average size was approximately 25 nm, with some being smaller (quantum dots) and interplanar spacing of 0.236 nm indicated a highly ordered crystalline structure. Moreover, the anticancer efficacy of the BVNPs was ascertained through in vitro assays against MCF-7 breast cancer cells, showing a dose-dependent cytotoxic effect. The findings of this study underscore the viability of hydrothermal synthesis in producing biologically active and structurally stable BVNPs, with a significant potential for anticancer activities., (© 2024. The Author(s).)