: In this study, the seroprevalence of Toxoplasma (T.) gondii and Bartonella (B.) henselae infection among stray cats in Daejeon City, Korea was surveyed. A total of seven samples were positive (7/118, 5.93%) for T. gondii including three samples from female cats (3/58, 5.2%) and four samples from male cats (4/60, 6.7%). There was no significant difference between the genders. A total 22 samples (22/118, 18.6%) were positive for B. henselae; nine were from females and 13 were from males. There was no significant difference between genders. Nineteen samples had a titer of 1 : 50, two samples had a titer of 1 : 100, and one sample had a titer of 1 : 200. The present study is the first to use serological tests to analyze B. henselae prevalence among stray cats in Korea.