The Roma midfielder completed 90 minutes in his country's lacklustre 1-0 defeat to Group D surprise package Costa Rica on Friday but sustained a calf problem.
De Rossi underwent tests at a hospital in Natal, where Italy take on Uruguay on Tuesday, and will have an MRI scan tomorrow, while a member of the team's medical staff heightened fears surrounding the 30-year-old's condition.
"There is something wrong," professor Enrico Castellacci is quoted by Italian media.
Italy head into their final group match level on three points alongside Uruguay, after both teams claimed wins that ultimately eliminated England and suffered shock losses that secured Costa Rica's shock progression to the last 16.
A draw for Cesare Prandelli's men would seal the second qualifying spot by virtue of a superior goal difference to Uruguay.