ABSTRACT The use of copper nanoparticles (CuNP) in the diet of broiler chickens has been studied as a potential alternative to antibiotic growth promoters. This study aimed to analyze the antimicrobial properties of CuNP in the feed and water of broiler chickens against Salmonella Enteritidis and to assess the intestinal integrity and toxicity of CuNP supplementation in their diet. The antimicrobial activity of CuNP against S. Enteritidis was tested in microplates to evaluate three water samples with different mineral compositions and in an in vitro digestibility model that simulated the three primary intestinal compartments of birds to assess feed samples. To evaluate in vivo intestinal integrity and toxicity, the birds were divided into four groups (30 birds per group): (1) basal diet (control); (2) basal diet + CuNP (100 ppm); (3) basal diet + enramycin (10 ppm); and (4) basal diet + CuNP (100 ppm) + enramycin (10 ppm). Intestinal samples were collected for histomorphometric evaluation and lactic acid bacteria count, while chest muscle and whole blood samples were collected to determine copper content. A significant reduction in the S. Enteritidis count was observed in both in vitro treatments (water and feed) with CuNP inclusion, compared to the control group. No significant differences histomorphometric measurements, weight gain, or total lactic acid bacterial counts were found compared to those in the control. These results demonstrate the effectiveness of CuNP in reducing the occurrence of S. Enteritidis and their non-interference with the intestinal integrity of broiler chickens, highlighting the potential of CuNP as an alternative antimicrobial agent in the poultry production chain.