Raccoons (Procyon lotor) live at high densities, often in close association with people, in urban areas in Ontario and have been implicated as potential reservoirs of numerous zoonotic disease agents. We collected 137 blood samples from 61 apparently healthy raccoons in a small area of Toronto, Ontario, from June to October 2007 as part of a longitudinal study to determine the seasonal patterns of seroprevalence of Francisella tularensis, avian influenza, and Leptospira. In addition, we collected 35 urine samples by cystocentesis from 23 animals to look for evidence of urinary shedding of Leptospira. All samples were serologically negative for F. tularensis and avian influenza. Nineteen of 61 animals (31%) were positive for Leptospira antibodies in one or more trapping periods. The seroprevalence of Leptospira increased from 5% in June to 38% in October. Of the 19 positive animals, 14 were seropositive for serogroup Grippotyphosa, 4 for serogroup Pomona, and 1 for both serogroups Australis and Grippotyphosa. Raccoons were seronegative to serovars representative of serogroups Autumnalis, Canicola, Icterohaemorrhagiae, and Sejroe. Only one urine sample was culture positive for Leptospira (2.9%). Although we found evidence that raccoons in this study were exposed to leptospires belonging to serogroup Grippotyphosa, likely serovar Grippotyphosa, during the summer and able to shed leptospires in urine, further work is required to determine the importance of raccoons as reservoirs of Leptospira in Ontario.