Nowadays, there are progressive advancements in the biomedical employment of nanotechnology in human and animal health. Today, the dangers of infectious animal diseases and the continuous application of traditional antibiotics resulted in the elevation of the activities of the drugs resistant genes and the failure of their control. Hence, it is necessary to prevent fungal and bacterial pathogens pollution and their toxicity in dairy animal products. Sixty samples were collected from dairy buffalo farms suffering from subclinical mastitis (20 Samples of each from subclinical mastitis milk, animal feeds, and bedding). The species of Staphylococcus are considered the most predominant species, and Staphylococcus aureus was the most prevalent bacterial isolate. While, yeast, especially Candida albicans, was the most dominant fungal isolate from different samples of dairy buffalo farms. Our antibiogram of S. aureus and Candida albicans showed a high resistance rate against several traditional antimicrobial agents. Therefore, searching for effective drug materials to overcome drug resistance is needed. Herein, ZnONPs and Curcumin are evaluated singly and in a composite to inhibit the growth of C. albicans and S. aureus. The inhibitory dose of ZnONPs for C. albicans and S. aureus were (600 and 600 μg /ml) and in Curcumin were (2% and 3%), respectively. However, the hybrid nanocomposite of ZnONPs (100 μg/ml) with Curcumin (0.25% and 1%) caused suppression of the growth of these pathogens. The nanocomposite of low levels of ZnONPs (100 μg /ml) with (0.25% curcumin) significantly increased the inhibitory effects against C. albicans and S. aureus. The molecular study to detect the efficiency of the treatment doses indicated that the exposure of C. albicans and S. aureus to a low dose of ZnONPs (100 µg/ml) did not affect the DNA expression of virulent genes. While the high dose (600 µg/ml) prevented the DNA expression. However, in the Curcumin treatments of isolates with low doses (0.25), the genes' DNA expression was not affected. While, the high dose (1%), removed the DNA band. All inhibition and removal of virulent genes' DNA of pathogens can be obtained by the combination of low levels of ZnONPs and Curcumin. Hence, it is concluded that the nanocomposites of ZnONPs and Curcumin caused significant effects against pathogens.