From 2005 to 2017, a total of 334 fresh fecal samples was obtained from 236 captive and free-ranging Grand Cayman iguanas (Cyclura lewisi) in a captive breeding and release program. One-hundred-sixteen samples were obtained from individual captive iguanas living in enclosures with natural substrate, 161 samples from captive iguanas living in elevated wire-bottom enclosures, and 57 samples from free-ranging wild iguanas. These samples were cultured to isolate subspecies of Salmonella enterica. as part of prerelease health evaluations, and to determine baseline health assessments of captive and wild populations of these iguanas. There was a 5.45% (95% confidence interval [CI]: 1.14–15.12) prevalence of S. enterica in iguanas housed on natural substrate (n = 51), 3.85% (CI: 1.42–8.19) in iguanas housed in elevated wire-bottom enclosures (n = 157), and 6.06% (CI: 0.74–20.23) in wild free-ranging iguanas (n = 38). These results demonstrate no significant difference (P = 0.73) in S. enterica prevalence among these housing conditions. The incidence of S. enterica from 2005 to 2017 in the population sampled was 4.19% (CI 3.10–5.29). Ten different serotypes of S. enterica were isolated from 14 iguanas. Salmonella enterica ser. Saintpaul was the most frequent isolate. Annual S. enterica prevalence was calculated for iguanas housed in different enclosure types, for free-roaming wild iguanas, and for all cultured iguanas. The highest yearly prevalence was 23.08% (CI: 5.04–53.81) in iguanas tested in 2007 (n = 21). No Salmonella enterica were cultured in 2005, 2008, 2009, 2010, 2011, 2012, and 2015. These results suggest that the shedding of S. enterica was not significantly different between housing types or between captive versus wild iguanas and therefore that release of captive iguanas did not significantly affect the health of the wild population or their exposure to S. enterica.