LaLiga leaders Real began their quest for a record-extending 15th European Cup with a last-gasp 1-0 home victory over Union Berlin last month, while Italian champions Napoli, who are currently third in Serie A, won 2-1 at Sporting Braga.
Tuesday's clash at the Diego Armando Maradona Stadium could be pivotal to both sides' aims of taking top spot in Group C.
"We'll play against one of the best teams in Italy. They did very well last year. It will be a competitive and evenly matched game as they have a high level," Ancelotti, who once managed Napoli, told a news conference.
"It's going to be the toughest game of the group stage for us. I don't want to say that we're used to it, but the shirt of this club weighs on us."
The match will also see Ancelotti return to Naples after he managed 73 games at the club from 2018 to 2019, leading the team in an unbeaten run in the Champions group stage before being sacked with the team in seventh place in Serie A.
"I had positive moments, it is a wonderful city. There were also less good moments, but I have the memory of a positive experience," Ancelotti said.
"Backtracking before a game like this doesn't make sense. When the relationship between a club and a coach doesn't have the right feeling, it's better to stop.
"I think it was the right decision for Napoli and for me, because two years later I came back to the best club in the world."
Real Madrid and Napoli last met in the Champions League in the 2016/17 round of 16 when the Spanish giants won both legs 3-1 to and went on to claim the title.
