This study introduces an environmentally friendly technique for copper nanoparticle synthesis utilizing Malpighia glabra fruit extract under the sonication treatment. The synthesis process and phenol red removal were optimized by a central composite full and response surface design. Highly pure and spherical-shaped copper nanoparticles with an average size of 22.5 nm were formed using 7.4 mL of Malpighia glabra fruit extract and 21.9 mM (AcO)₂Cu. Additionally, the extract-mediated nanoparticles opposed the negative charges with a zeta potential of -11.8 mV and high stability of 30 days storage time. The sonication-assisted nanoparticles exhibited the highest inhibition against Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus), MCF7 human breast cancer cells, and Fusarium solani with 50% inhibition concentrations reaching 12, 0.82, and 80 ppm, respectively. Additionally, the green-synthesized nanomaterials functioned as an effective catalyst to remove phenol red. A conversion of 97% after a 540 seconds reaction was determined on 10 ppm phenol red with the presence of 21.5 ppm copper nanoparticles and 51.8 mM NaBH₄. This research highlights the potential of Malpighia glabra fruit extract in the sustainable production of copper nanoparticles, with promising applications in biomedicine, agriculture, and environmental remediation.