Due to nanoscale properties that can be fine-tuned to treat different diseases, putting nanotechnology into the field of biology has become a popular way to study medicine. Here, we synthesized zinc oxide nanoparticles using the seed extract of Artocarpus hirsutus. A qualitative phytochemical study of the seed extract confirmed the presence of flavonoids and proteins, which encouraged its use in the antibacterial study. The synthesized nanoparticles are characterized by XRD (X-ray diffraction), SEM (scanning electron microscopy), EDAX (energy dispersive X-ray analysis), UV–visible spectroscopy, and FTIR (Fourier transform infrared spectroscopy). XRD analysis revealed the particles are crystalline in nature, with an average crystal size of about 25 nm. SEM spectroscopic studies revealed the particles were rod- and spherical-shaped, and the EDAX spectrum confirms the presence of zinc and oxygen in the synthesized nanoparticles. The UV–vis spectrum revealed absorption spectra at 266 nm and 365 nm that confirmed the presence of ZnO nanoparticles. The presence of proteins and amide groups acts as a reducing and stabilizing agent, which was confirmed from the FTIR spectrum. Antibacterial studies against two gastro-intestinal bacteria (Enterobacter aerogenes and Listeria monocytogenes) were conducted using the well-diffusion method and scanning electron microscopy. The impact of zinc oxide nanoparticles on the HT-29 colon cancer cell line was investigated. MTT, DCFDA labeling, DNA fragmentation tests, flow cytometry, and gene expression on Bcl-2 were used to reveal that ZnO nanoparticles had remarkable cytotoxicity activity against the HT-29 cell line. The results highlight the efficacy of zinc oxide nanoparticles in penetrating gram-negative bacteria more than gram positive bacteria and their efficient anticancer activity on HT-29 colon cancer cells. This research reports the synthesis of zinc oxide nanoparticles for the first time using Artocarpus hirsutus seed extract and also the evaluation of their antibacterial and anticancer activity on HT colon cancer cells.