O'Brien informed Racing Victoria stewards the Irish St Leger winner Order Of St George and Mackinnon Stakes candidate Ol' Man River would not travel to Melbourne.
O'Brien's decision leaves the stable with three Melbourne spring contenders with Cox Plate entry Highland Reel and Melbourne Cup runners Kingfisher and Bondi Beach all likely to travel to Australia.
Prominent Melbourne owner Lloyd Williams bought shares in Order Of St George, Kingfisher and Bondi Beach last month.
Order Of St George was fourth in the Melbourne Cup order of entry when first acceptances were taken on Tuesday.
Bondi Beach is eighth in line for a Melbourne Cup start while Order Of St George's withdrawal pushes Kingfisher (28th) another step closer to a run at Flemington on November 3.
Highland Reel is an $8 chance to win the Cox Plate while Bondi Beach and Kingfisher are at $17 and $21 respectively in Sportsbet's latest Melbourne Cup market.