Rodents and other small wild mammals are often considered to be pests and vectors for disease in zoos that house small populations of valuable threatened and endangered animals. In 2005, three nonhuman primates at a drive-through zoo in Oregon, US, acquired tularemia from an unknown source. Due to an abundance of California ground squirrels (Spermophilus beecheyi) on zoo grounds, we instituted serosurveillance of this species from July through September 2008 to determine the prevalence of antibodies against pathogens considered to be potentially transmissible to collection animals. Serologic testing was performed for Francisella tularensis; Leptospira interrogans serovars Canicola, Grippotyphosa, Hardjo, Icterohemorrhagiae, and Pomona; Toxoplasma gondii; and Yersinia pestis. All squirrels were seronegative for Yersinia pestis (0%; 0/45) and Toxoplasma gondii (0%; 0/20); there was a prevalence of 2% (1/45) for Francisella tularensis antibodies and 57% (24/42) were positive for various Leptospira serovars. Although it remains unclear whether ground squirrels present a significant risk for transmission of disease to zoo animals, vaccination of high-risk zoo animals against leptospirosis warrants consideration. Beyond this, continued vigilance and persistence with various forms of pest control may reduce the likelihood of disease transmission from wildlife hosts to animals in human care.