Mayweather, who is moving up to 154lbs for the first time, stood nose-to-nose with his opponent, bumping him with his chest and calling him a "pussy".
De la Hoya, 34, remained calm, insisting he would make Mayweather pay once they got into the ring, with the world's best pound-for-pound fighter tag at stake.
"When he's talking, it's just more fuel, baby," De la Hoya said through gritted teeth.
"I respect Floyd as a fighter but come 5 May, he's going to be hurting for weeks."
The hotly-anticipated clash has already sold out for a Nevada-record gate of $19m.
A record 1,200 closed circuit showings are expected for the fight, to be broadcast to 176 countries.
De la Hoya has been trained for the last six years by the challenger's father, Floyd Mayweather Sr, but will have veteran Freddie Roach in his corner for the big fight.
But Mayweather mocked De la Hoya and said he would back up his bravado with hard cash.
He added: "I'll bet you $1m, $2m, $3m I'll beat you. Every dime in my bank account, right now."