They say a Markham resident is the region's first "probable" human case of the virus this year.
There have been two other reported cases since 2008.
The virus is transmitted to humans through infected mosquitos, but many cases go undetected.
York's medical officer of health says West Nile activity is on the rise across the province and is urging residents to protect themselves against bug bites.
Health officials in nearby Toronto have already found two people in the city who may have the virus.
Saskatchewan also reported its first human case earlier this month.