In August 2015, a gopher tortoise (Gopherus polyphemus) mortality event was documented on a 40-ha area of Lake Louisa State Park, Lake County, Florida, US. To quantify the extent of the die-off and the seroprevalence of Mycoplasma agassizii and Mycoplasma testudineum, two causative agents of mycoplasmal upper respiratory tract disease (URTD), we conducted a tortoise shell survey on 25 and 26 August 2015 and collected blood samples from live tortoises on 2–4 September 2015 and 1–13 August 2016 within the area of documented mortality. We discovered 94 shells and measured the degree of disarticulation to estimate time since mortality. Results indicated that most mortalities likely occurred more than 3 yr before discovery. Of the 42 blood samples collected in 2016, 31% (n=13) tested positive, 17% (n=7) were suspect, and 52% (n=22) were negative for M. agassizii antibodies; all blood samples were negative for M. testudineum. Sixty-nine percent (9/13) of seropositive tortoises exhibited clinical signs of URTD, and seropositive individuals were more likely to express clinical signs than seronegative tortoises. However, 32% (7/22) of seronegative individuals also exhibited some clinical signs, including naris and eye abnormalities. We suggest additional research to determine causality of this mortality event, as well as examine risks associated with its spread.