The South American side kick-off their Group B campaign again Ange Postecoglou's unfancied team at Cuiaba's Arena Pantanal on June 13 before facing Spain and the Netherlands.
And despite the Socceroos having been written off as likely whipping boys, Sampaoli has claimed to be wary of a match his players cannot afford to lose if they are to progress to the knockout stages.
"Always the first opponent is the most complicated," he said.
"In a tournament where you need to quickly (gain points), there will be nervousness, beyond what Australia can offer.
"That will be the hardest game... anxiety becomes secondary when you have so much work to do. (This is) no time to think about how many days are left to face Australia or who are the players going out. We must continue to strengthen the group."
While Brazil and Argentina are favourites to win on the 'home soil' of South America, Sampaoli's talented side are also being talked up as potential dark horses, with the weight of domestic expectations hanging over his players.
"The enthusiasm of the people requires us to prepare in the best way," he said.
"(We will) try... all through the preparation for the players (to) arrive in the best way to confront many rivals (with different qualities). Beyond the position (the) team will finish in our group, it is important that Chile does not lose shape against such prestigious opponents.
"My responsibility is to prepare the squad to face a very competitive group. I must try (to make sure) that players who came in (at) different point of preparation are (at their) best.
"Our priority is to work, (the intensity of) which is negotiable. Planning is scheduled in a certain way, but here athletes have to (combine) the mental with the physical (and) with the football. So, that's what we are (at)."
