The Super Eagles made four changes to the team that beat Poland 1-0 in Wroclaw on Friday, with the quartet of Tyronne Ebuehi, Chidozie Awaziem, Ogenyi Onazi and Ahmed Musa introduced to the line-up.
However, the changes served to undermine Nigeria's intensity, and their showing was a far cry from the high pressing and dynamism of recent fixtures.
Mitrovic might have opened his account in the first half, but his effort was ruled out despite his header appearing to cross the line before Francis Uzoho desperately scrambled to gather.
It was the second time in four days the Eagles had a controversial goalline decision go in their favour.
Uzoho did well to save from Mitrovic, Dusan Tadic and Branislav Ivanovic, and the youngster looked poised to preserve a third successive clean sheet in the green-and-white of Nigeria until the on-loan Fulham forward's late intervention.
Mitrovic broke the deadlock in the 68th minute with a composed finish, and added a second - exploiting some poor defending on behalf of the West Africans - in the 81st minute as Nigeria pressed forward in search of an equaliser.
Nigeria's best chance fell to Victor Moses, as the Chelsea wing-back forced Vladimir Stojkovic into a smart save at the death
The defeat was only the second defeat of Gernot Rohr's tenure, and the German tactician will surely have learned a lot from the performance of some of his peripheral players in their loss against the Eastern Europeans.