She started her career as a stage actress at the Liverpool Playhouse. Her screen debut was in A Taste of Honey (1961), the groundbreaking movie of the British realist tradition. Other performances included The Leather Boys (1962), Girl with Green Eyes (1963), The Knack …and How to Get It (1965), Doctor Zhivago (1965), The Trap (1966), Smashing Time (1967), The Bed Sitting Room (1969) and The 'Human' Factor (1975).

In 1972, she was a member of the jury at the 22nd Berlin International Film Festival.

She was an icon of 1960s Britain, and was popularised again in the 1980s by her appearance in Carla Lane's Bread.

In 1989 she was Mrs Deakin in Resurrected, appearing with David Thewlis and Tom Bell.

She was last seen in the film Being Julia (2005) and in "The Sittaford Mystery", a 2006 episode of Marple.

In 2008 she was reported to be starring in a number of films due for release including a mystery thriller The Hideout, a romantic drama Broken Lines, Telstar, about the tragic life of legendary British record producer Joe Meek, and The Calling, in which she plays a nun, Sister Gertrude.

Clips from her performance in The Leather Boys appeared in The Smiths' music video for the single, "Girlfriend in a Coma", in 1987. She is also mentioned in the Franz Ferdinand song "L. Wells" and the Television Personalities song "Favourite Films".In 1999 she was featured on "This Is Your Life"

Tushingham married the photographer Terry Bicknell in 1962. They had two daughters, Dodonna and Aisha Bicknell. In 1981 she married Iraqi cinematographer Ousama Rawi, spending eight years in Canada with him. She now divides her time between Germany and London, with her partner since the mid-1990s, writer Hans-Heinrich Ziemann.

In April 2005, at the age of 33, her daughter Aisha Bicknell was diagnosed as suffering from breast cancer. Tushingham became an activist for breast cancer health and support. She and her daughter support Cancer Research UK's Relay for Life and have given a number of interviews to raise breast cancer awareness.

In July 2009 she was bestowed with an Honorary Fellowship by Liverpool John Moores University for "outstanding and sustained contributions to the Performing Arts".