Wilkof is from Canton, Ohio, a town of approximately 80,000. His father ran a steel service center. Wilkof was the middle child of Anne Louise and Darwin Wilkof. He has two brothers, Todd and Robert. He graduated from the University of Cincinnati in 1972 and studied acting with Austin Pendleton in New York City.
After graduating from UC, Wilkof wrote and starred in a comedy revue called The Present Tense in 1977 at the Park Royal Theatre in New York City. Wilkof moved to Los Angeles and took small film and television roles. His big break came in 1982 with the leading role as Seymour in the original Off-Broadway production of Little Shop of Horrors, and he also recorded the cast album.
Wilkof has appeared in numerous television series, and films. He directed the film No Pay, Nudity with a cast that stars Nathan Lane, Gabriel Byrne, and Frances Conroy.
He is the recipient of an Obie Award, a SAG Award, and has been nominated for a Tony Award and three Drama Desk Awards.
Wilkof lives in Brooklyn, New York with his wife, painter Connie Grappo. They have one child, a daughter.