He was on board NASA's Columbia space shuttle flight in February 1996 to test the tethered satellite system (the Italian satellite was lost when the tether broke). He also flew on the Endeavour mission in April 2001 to deliver the robotic arm, Canadarm 2, to the International Space Station. He has logged 681 hours in space.

Guidoni was involved in the development of the tethered satellite system when selected as an astronaut by the Agenzia Spaziale Italiana (Italian Space Agency) in 1989. He became a European Space Agency astronaut in 1998 and began training at the Johnson Space Center in 1996. He retired from active astronaut status in June 2004.