<p>The Florida spotted skunk (<i>Spilogale putorius ambarvalis</i>) is disjunct from other eastern spotted skunk (<i>S. putorius</i>) subspecies and has rarely been studied. Declines in eastern spotted skunk populations elsewhere underscore the importance of better understanding the Florida subspecies’ ecology and resource use, including its potential impacts as a nest predator of imperiled ground-nesting birds. The objective of our study was to gather baseline information on Florida spotted skunk diet in the dry prairie ecosystem of Central Florida. We conducted a stable isotope analysis using hair samples from 39 skunks and compared these with 106 samples from potential food items in dry prairie in 2016 and 2017 at Three Lakes Wildlife Management Area in Osceola County, Florida. Our resultant Bayesian mixing model indicated that the most prevalent food items in the Florida spotted skunk diet were millipedes (Diplopoda; ~ 42% of the diet) and a group consisting of amphibians and reptiles (~ 16%). Less prevalent were the groups including ground-nesting birds—one group containing granivorous or herbivorous species (~ 15%) and another containing insectivorous species (~ 13%). Our study provides evidence that Florida spotted skunks in the dry prairie ecosystem likely have a generalist diet, like spotted skunks outside of Florida, as no food item composed a majority (&gt; 50%) of their diet. This study provides the first insights into the diet of free-ranging Florida spotted skunks and fills a key knowledge gap in the ecology of this important predator of the Florida dry prairie ecosystem.</p>