Mathyssen was educated at the University of Western Ontario from 1970 to 1975, receiving a Bachelor of Arts degree in English and Drama, and a Bachelor of Education degree. She taught English at Saunders Secondary School in London, Ontario, until 2006 except for a five-year break between 1990 and 1995 when she was an MPP. She served on the District 11 Ontario Secondary School Teachers' Federation Political Action Committee. She was a vocal opponent of the Invasion of Iraq in 2003 and participated in many local peace rallies. Mathyssen was a member of the "Ontario Health Coalition". She was president of the Middlesex NDP riding association from 1989 to 1990.