In urban rivers, many of which are used for drinking water production, viruses encounter a range of particulate, colloidal, and dissolved organic and inorganic compounds. To date, the impact of environmental organic matter on virus persistence in the environment has received little attention. In the present study, fresh water was fractioned to separate particulate natural organic matter from dissolved forms. Each fraction was tested for its ability to promote coxsackievirus B5 resistance to heat inactivation. Our results demonstrate that, at natural concentrations, environmental waters contain particulate or dissolved compounds that are able to protect viruses from heat. We also show that hydrophobic compounds promote an efficient protection against heat inactivation. This study suggests that local conditions encountered by viruses in the environment could greatly impact their persistence.