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The eco-friendly production of palladium nanoparticles (PdNPs) by Prunus × yedoensis tree leaf extract was studied for the first time. Initial confirmation of PdNP production was confirmed by a color change from light yellow to dark brown. The optimization parameters show that pH 7, 40:5 Pd (II): leaf extract, 3 mM Pd (II), and a 30 min timeframe are essential for the production of PdNPs. The UV–Visible spectra (421 nm), X-ray diffraction peaks (2θ=42.5°), and Fourier transform infrared spectroscopy confirmed the presence of PdNPs and phytochemicals. Morphological structure analysis using high-resolution transmission electron microscopy shows that the PdNPs are mostly spherical in shape with rough edges with a size of 50–150 nm. Among all the gram-positive and gram-negative bacteria tested, Bacillus subtilis (6 mm) and Pseudomonas aeruginosa (5 mm) showed a zone of inhibition against the synthesized PdNPs at 200 μg/well.