Safa announced the withdrawal of the two players in a statement, with Dikgacoi having to miss out due to a calf ligament tear which he suffered during Crystal Palace's 1-0 win over Watford in the Championship playoff final on Monday.
Masilela will undergo surgery for a long-standing knee injury, which he has been playing with throughout the season.
Siboniso Gaxa's injury was reported on Tuesday, with the right-back limping out of the Nedbank Cup final on Saturday. Siyanda Xulu is the likely replacement for Gaxa, but the FC Rostov defender has only just returned from a long-term injury in Russia.
Ricardo Nunes, the Slovakia-based full-back will replace Masilela but with South Africa already missing Morgan Gould, Siyabonga Sangweni and Thabo Matlaba their defensive reserves are becoming slim.
Read more on: South Africa's latest squad selection
“We have really had some bad luck with defenders lately missing out due to injury; this is really a big blow for us in that department," Bafana Bafana coach Gordon Igesund said.
“Already, we are without Morgan Gould, Siyabonga Sangweni, Thabo Matlaba, and now Masilela.
Siyanda Xulu recently returned from an injury, but they say when one door closes the other opens, so I believe this is a chance for the players in the squad to raise their hands and also help their country through these difficult times.”
Igesund expressed his happiness with Crystal Palace's promotion into the Premier League, but has not yet thought of a replacement for Dikgacoi and is hoping he can recover in time.
“I am very happy for KG for the promotion to the English Premiership, but it is just unfortunate he won’t be with us. He is a good player and I believe he deserves to be playing at the highest level which will in turn help Bafana," Igesund said.
“We are hoping for a speedy recovery for him. I still have to apply my mind as to whether to call in a replacement, but if I do it will be a player based locally. For now I want to sleep on it as I don’t want to make any quick decisions.”