Dusseldorp attended Geelong Grammar School, graduating in 1990, and the University of New South Wales, where she majored in theatre and film for two years. She then went on to study at the Victorian College of the Arts in Melbourne. She is the granddaughter of Dutch Engineer Dick Dusseldorp, the founder of Lend Lease Corporation.

She won the award for Best Female Actor in a Supporting Role in a Play in the 2009 Helpmann Awards for her performance as Queen Margaret in Benedict Andrews' The War of the Roses with the Sydney Theatre Company. Dusseldorp returned to the stage in 2016 in the premiere of Benedict Andrews' play Gloria with the Griffin Theatre Company. In November 2017, Dusseldorp and her husband, Ben Winspear, will play the couple in Joanna Murray-Smith's stage adaptation of Scenes from a Marriage for the Queensland Theatre Company.

Dusseldorp played the character of Janet King in the 2011 ABC1 drama series Crownies, a role she reprised in the 2014 spin-off Janet King. She is also known for the telemovie series BlackJack (2003–2007) as Sam Lawson and the 2012 Jack Irish telemovie series as Linda Hillier. She has appeared in guest roles on television series such as All Saints. She has a starring role in the ongoing series A Place to Call Home, which commenced in 2013.