The presence of large numbers of free-ranging feral cats (Felis catus) has raised concern in terms of both native species predation and potential disease transmission in Hawai‘i. A disease of particular concern is toxoplasmosis, caused by Toxoplasma gondii, a zoonotic protozoan parasite. We tested soil samples and cat fecal samples from cat colonies from an urban university campus and a natural, coastal ecosystem for T. gondii oocysts using standard molecular procedures. Soil and fecal samples were collected from cat colony sites at the University of Hawai‘i at Mānoa (UHM), and additional fecal samples were collected from cats trapped within Ka‘ena Point Natural Area Reserve (KPNAR). Toxoplasma gondii DNA was detected in 5% (3 of 60) of fecal samples from UHM, but no T. gondii DNA was detected from soil samples. At KPNAR, 22.2% (2 of 9) of fecal samples were positive for T. gondii DNA. Presence of T. gondii at the university study sites suggests that cat colonies may be a potential health hazard for landscaping personnel, students, staff, and visitors. Likewise, presence of T. gondii at KPNAR, and potentially other coastal habitat(s) for ground-nesting seabirds or marine mammals, also suggests a disease risk and should be considered when managing those areas. Toxoplasmosis is a growing concern to both people and wildlife, and further work is needed to determine pathways of transmission both within and between terrestrial and marine ecosystems of Hawai‘i.