This paper reports the green synthesis of Copper Oxide nanoparticles (CuO NPs) using Aquilaria malaccensis (agarwood) leaf extract. The main objective of this study was to evaluate the potential of using A. malaccensis leaf extract as a biogenic medium to generate CuO NPs with antimicrobial potential. The procedure employed was to add 5 mM copper sulfate (CuSO4.5H2O) as the precursor to A. malaccensis leaf extract to study the generation of CuO NPs under different incubation conditions such as methods of crude extract preparation, precursor concentration and incubation temperature. The results demonstrated that the boiled leaf extract reacted with 5 mM CuSO4.5H2O at pH 6 and incubated under non-shaking conditions at 70 °C, resulting in a high rate of CuO NPs formation and depicting a UV absorbance peak of 430 nm. Green synthesized CuO NPs were characterized using field emission scanning electron microscopy (FESEM) and energydispersive X-ray spectroscopy (EDX), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and transmission electron microscopy (TEM). FESEM and TEM revealed that the nanoparticles are mainly spherical, ranging from 6 to 32 nm. Antimicrobial studies showed that 20 µL and 40 µL of 70 µg/µL CuO NPs displayed potent inhibition towards Gram-positive bacteria Bacillus subtilis, with the average zone of inhibition measuring 24.43 ± 0.10 mm and 27.31 ± 0.13 mm, respectively.