From 2008 to 2017, blood samples from 280 and fecal samples from 149 stray cats in Gwangju, South Korea, were examined for feline immunodeficiency virus (FIV), feline leukemia virus (FeLV), Dirofilaria immitis, and Giardia infections using commercial diagnostic tests. Overall, the combined prevalence of FeLV, FIV, D. immitis, and Giardia was 8.6%, 1.4%, 0.4%, and 2.0%, respectively. FeLV exhibited the highest prevalence rate among the 4 pathogens surveyed, both in the 2008?2009 (9.6%) and 2015?2017 (6.3%) surveys. The results of the feline Giardia study represent the first prevalence report of Giardia infection among stray cats in Korea.