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The application of nanoparticles rose steeply in the last decade, where they have become a common ingredient used in processed human food, improving food properties such as shelf life and appearance. Nanoparticles have also attracted considerable interest to the livestock industry, due to their efficacy in intestinal pathogen control, with the regulatory and consumer driven push for the removal of antibiotic growth promoters. The influence of selenium (Se) nanoparticles was investigated on a diverse and mature broiler caecal microbiota using in vitroculturing and 16S rRNA gene sequencing methods for microbiota characterisation. Caecal microbiota was collected from 4 traditionally grown heritage roosters and grown for 48 h, in the presence and absence of Se nanoparticles, with 2 technical replicates each. The effect of rooster as a biological variable strongly overpowered the effects of nano-Se in the media, resulting in moderate effects on the structure and diversity of the caecal microbial community. However the nanoparticles showed a significant reduction (P < 0.05) in the abundance of an emerging poultry pathogen, Enterococcus cecorumidentical operational taxonomic units (OTU), which could be of notable interest in poultry production for targeted E. cecorumcontrol without significant disturbance to the total microbial community.