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Coliforms and <i>Staphylococcus</i> spp. infections are the leading causes of bovine mastitis. Despite extensive research and development in antibiotics, they have remained inadequately effective in treating bovine mastitis induced by multiple pathogen infection. In the present study, we showed the protective effect of <i>Zophobas morio</i> (<i>Z. morio</i>) hemolymph on bovine mammary epithelial cells against bacterial infection. <i>Z. morio</i> hemolymph directly kills both Gram-positive and Gram-negative bacteria through membrane permeation and prevents the adhesion of <i>E. coli</i> or the clinically isolated <i>S. simulans</i> strain to bovine mammary epithelial (MAC-T) cells. In addition, <i>Z. morio</i> hemolymph downregulates the expression of nucleotide-binding oligomerization domain (NOD)-like receptor family member pyrin domain-containing protein 3 (NLRP3), caspase-1, and NLRP6, as well as inhibits the secretion of interleukin-1β (IL-1β) and IL-18, which attenuates <i>E. coli</i> or <i>S. simulans</i>-induced pyroptosis. Overall, our results suggest the potential role of <i>Z. morio</i> hemolymph as a novel therapeutic candidate for bovine mastitis.