This work reports the optimization, synthesis, characterization, anticancer, and antibacterial activity of the Trichoderma-β-D-glucan‑zinc oxide nanoparticles (T-β-D-glu-ZnO NPs). Firstly, the T-ZnO NPs was synthesized using the fungal mycellial water extract (FWME) derived from T. harzianum (SKCGW009) under the optimized condition of extract concentration (5.99 mL), temperature (43.11 °C), pH (8) and time (69.04 h). The successful conjugation of T-ZnO NPs with β-D-glucan (T-β-D-glu-ZnO NPs) was confirmed by PACE and FTIR. The XRD, UHR SEM, and TEM EDS results pointed the spherical shape of NPs with the mean size of 30.34 nm. Further, the XPS survey scan and high-resolution fitting of Zn2p results also claimed the successful formation of the T-β-D-glu-ZnO NPs. Cytotoxicity results indicated that the NPs were not toxic to NIH3T3 cells, while exhibited the dose-dependent inhibitory effect to human pulmonary carcinoma A549 cells. The IC of T-ZnO NPs and T-β-D-glu-ZnO NPs against A549 cells was 158 and 56.25 μg.mL, respectively, which was also verified by fluorescent cytochemistry. Annexin V-FITC staining results indicated the presence of apoptotic cells in the NPs treated A549 cells, which was not seen in the non-treated control A549 cells. Interestingly, the number of necrosis cells was higher in the T-ZnO NPs (3.38%) comparing to T-β-D-glu -ZnO NPs (0.07%). The early or late apoptosis was found higher in the cells treated T-β-D-glu -ZnO NPs (6.43%) comparing with T-ZnO NPs (4%). These results indicated that T-ZnO NPs and T-β-D-glu-ZnO NPs induced the cancer cell death through necrosis and apoptosis pathway, respectively. The antibacterial results indicated that the NPs treatment were significantly inhibited the growth of the Staphylococcus aureus inside of roundworm and enhanced growth of roundworm. Overall, anticancer and in vitro, in vivo antibacterial studies proved the high caliber of T-β-D-glu-ZnO NPs for the further pharmaceutical evaluation.