With former Lions of the North mentor Allan Freese at the helm, Eagles succumbed to a 2-1 defeat at the Harry Gwala Stadium.
Goals from Petrus Ngebo and Phoka Mofokeng sealed victory for the visiting side, who kept intact their perfect record having won their opening day fixture against Jomo Cosmos.
Meanwhile, Stellenbosch were held by second-tier newbies Uthongathi whilst Real Kings and Witbank Spurs were unable to be separated.
Mbombela United recovered from an opening day defeat to Black Leopards by putting three past Super Eagles in the Free State.
Cape Town All Stars 1-0 Richards Bay
Real Kings 1-1 Witbank Spurs
Stellenbosch 1-1 Uthongathi
Royal Eagles 1-2 Highlands Park
Super Eagles 1-3 Mbombela
Jomo Cosmos vs AmaTuks
Mthatha Bucks vs TTM
Black Leopards vs Ubuntu Cape Town