Synthetic synthesis of nanoparticles is harmful to human and environment due to pollution. The use of harmful chemicals in synthetic method has avoided in the biosynthesis. To overcome the hurdles in synthesis of nanoparticles, the biogenic synthesis of CuO nanoparticles has been done with Plectranthus amboinicus leaf extracts. The biosynthesis provides the enhanced efficiency compared to synthetic methods. It establishes the environmentally friendly and pollution-free approach. The powder X-ray diffraction (XRD) pattern exhibited a crystalline nature with the crystallite size in 20–35 nm. The Fourier-Transform InfraRed (FTIR) spectrum explicated the Cu and O metal oxide bonds. The UV–visible analysis emphasized the stability of CuO nanoparticles with the inference of the Surface Plasmon Resonance band at 398 nm. The photoluminescence study displayed the bluish–green emission nature peak at 491 nm. The particle size analyzer manifested the synthesized CuO nanoparticles majority in 5–40 nm. The field-emission scanning electron micrographs (FESEM) and high-resolution transmission electron micrographs (HRTEM) exposed the spherical, circular-shaped nanoparticles with size in 5–30 nm range. The energy dispersive X-ray diffraction (EDAX) spectrum and mapping proved the CuO nanoparticles formation. The antimicrobial activity reported the potent nature of formed CuO nanoparticles on bacteria and fungi. The antioxidant activity identified the effective free-radical scavenging activity nature at 95% in 80 μg/mL and IC50 value at 40.10 μg/mL concentration. The anti-inflammatory activity assessed the efficient nature of CuO nanoparticles in protein denaturation of Egg albumin at 92% in 500 μg/mL and IC50 value at 243.08 μg/mL concentration. The anti-diabetic activity established the effective inhibition on α-Amylase at 94% in 500 μg/mL and IC50 value at 317.19 μg/mL concentration. The anti-larvicidal activity manifested the potent inhibition on Anopheles subpictus mosquito larvae within 24 h in 100 ppm concentration and which exhibited low LC50 value.