Development of efficient methods for treating microbial infections, cancer, and toxic organic dyes is a serious challenge in medical sciences. The purpose of this study is to synthesize CuO-NPs using T. parthenium extract and to evaluate its anticancer, antimicrobial, and catalytic activity. CuO-NPs were characterized by UV-Vis, XRD, FTIR, FESEM, and EDX. UV-Vis spectra exhibited surface plasmonic resonance at 298 nm of synthesized CuO-NPs. The synthesized CuO-NPs were pure, predominantly spherical with mean size of 16 nm. FTIR confirmed that CuO-NPs were reducted and stabilized with the biomolecules present in the T. parthenium extract. CuO-NPs indicated excellent degradation activity for the industrial dyes, i.e., MO (96.6% removal in 400s), Rh B (98.3% removal in 400s), MB (98.7% removal in 400s) and CR (99.6% removal in 180s). CuO-NPs showed excellent inhibition against selected microorganisms, especially E.coli and C. albicans. CuO-NPs have also shown good anticancer activity against A549, Hela, and MCF7 cancer cell lines (IC50 = 65.0, 57.4, and 71.8 µg/mL, respectively) while negligible cytotoxic effects were observed on L929 (IC50 = 226.1 µg/mL). The results proposed that synthesized CuO-NPs can be considered as a suitable candidate for biomedical, pharmaceutical, and environmental applications.