"Iniesta will miss tomorrow's game against Villarreal with an injury to his left knee," Barcelona said in a statement.
Neymar, who missed the opening day victory at home to Elche through an ankle injury, has been passed fit.
"I am hoping that the team begins to settle which is something that will happen with games," Luis Enrique, who is starting his first season as Barca coach, told a news conference on Saturday.
"We are looking sharp after a week where we have trained well. We know our rival (Villarreal) well and they are a strong team in the league. They had a good preseason in terms of performance and results.
"The game will not be easy for us but we are prepared and at the moment each match is a test for us. All teams with a new coach need time to adapt but that is something that I am not asking for."