<p>Similar to other herbivores, healthy giraffes (<i>Giraffa camelopardalis</i>) rely on a variety of symbiotic microorganisms in their digestive systems to break down cellulose and hemicellulose. In this study, we investigate the impact that external stimuli might have on the faecal prokaryote composition of healthy, free-roaming giraffes. Faecal samples were collected from six male and seven female giraffe individuals, over a 2-year period, during the wet and dry seasons, from six locations within the Free State Province, South Africa. Giraffe populations were exposed to one of two feeding practices which included provision of supplemental feed or only naturally available vegetation. Seventeen (17) different prokaryotic phyla, consisting of 8370 amplicon sequence variants (ASVs), were identified from the 13&#xa0;healthy, adult, free-roaming giraffes included in the study. Overall, the bacterial phyla with the largest relative abundance included Fusobacteria (22%), followed by Lentisphaera (17%) and Cyanobacteria (16%), which included 21 dominant prokaryotic ASVs. The relative abundance of Ruminococcaceae UCG 014 and <i>Treponema 2</i> were found to be significantly (<i>P</i> &lt; 0.05) higher and <i>Escherichia</i> / <i>Shigella</i>, <i>Romboutsia</i> and <i>Ruminococcus 1</i> significantly lower for giraffes receiving supplemental feed compared to natural available vegetation. This is the first study to investigate the composition of the faecal prokaryotic communities of healthy, free-roaming giraffes. The analysis of faecal prokaryotes contributes to the development of non-invasive methods for assessing the nutritional status and identifying health issues in giraffe populations. Ultimately, such advances are beneficial towards the larger-scale conservation, determining nutritional needs and management of other sensitive wildlife species, as well.</p>