AC Milan captain Montolivo was injured in the early stages of the friendly at Fulham's Craven Cottage following a crunching tackle from Ireland defender Alex Pearce yesterday.
Skippering Italy on the night he needed several minutes of treatment and was seen mouthing "it's broken" before eventually being carried off.
"Montolivo has what we suspected, a broken tibia," Italy physician Enrico Castellacci said.
"I haven't seen the X-ray yet to evaluate the extent of it but for a broken bone you can expect surgery."
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To make matters worse for Italian coach Cesare Prandelli, he also saw Fiorentina midfielder Alberto Aquilani, who had come on for Montolivo, forced off with an injury before half-time.
Prandelli, whose side kick off their World Cup campaign against England on June 14, has yet to prune his provisional 30-man squad down to a final group of 23 and he will now have to decide who will replace Montolivo.
In Rotterdam, Holland continued their build-up to the finals with a 1-0 win over fellow Brazil-bound Ghana, Manchester United striker Robin van Persie's fifth minute goal proving decisive.