The pair had scaled one of the entry gates to the Colosseum - a climb of around four metres - in an apparent attempt to see the site up close after dark.
But the stunt ended badly, with both of the tourists injured from the four-metre drop. One suffered only bruising, but the other has been left with a fractured pelvis, Il Corriere di Roma reported, identifying the tourists as Brazilians aged 30 and 32.
Friends of the duo called an ambulance, and after emergency care had been administered, police arrived at the scene shortly before 3am on Sunday. Both will face charges for trespassing, and the man with the fractured pelvis will likely remain in hospital for several weeks, though his companion has already been released.
They were unable to give a reason for the act, but it is not the first time foreign visitors have attempted to get a nighttime visit of the ancient amphitheatre.
Two Germans scaled the 45-metre high structure in August last year, publishing a video of their escapade which led to questions over security measures in place at Italy's monuments.
In light of recent terror attacks in Germany, France and Belgium, Italy's terror alert is at 'level 2' – the highest level possible in the absence of a direct attack. Security efforts have been ramped up at the country's cultural heritage site with increased surveillance and police patrols.
According to Il Messaggero, staff at the Colosseum had yet more trouble to deal with when returning to work on Monday morning, in the form of graffiti one of the ancient columns.
The word 'morte' (death) was painted in black on a column near the entrance site, before staff covered it with a cloth to work on an assessment of the damage.