Ireland welcome Gibraltar to the Aviva Stadium in Dublin on Saturday for a Group D encounter in which they will be heavily favoured to claim a comfortable win against a side for whom last month's 7-0 defeat to Poland represented their first competitive game.
O'Neill will be without two key players in the form of Everton duo Seamus Coleman and James McCarthy.
Full-back Coleman has not played since Everton's UEFA Europa League clash with Wolfsburg on September 18 due to a head injury, while midfielder McCarthy has a strained quad muscle.
Goalkeeper Shay Given will also miss out with a hip complaint, but confidence should be high among the hosts, especially after Ireland displayed good character to negotiate a tricky away trip to Georgia and earn a 2-1 win in their group opener.
But former Celtic and Aston Villa manager O'Neill is refusing to take anything for granted.
"That's exactly the message to the players," O'Neill said. "You don't want to be taking your eye off the ball. This is the important game for us.
"This is a game we have to win. Obviously, we have to win the game. But Poland found it difficult. They scored a host of goals towards the end of the game.
"They [Poland] said they'd found it difficult and I wasn't sure what they meant until I watched the game. Honestly, Gibraltar did cause a problem or two.
"So this idea that it's just all over? We're expected to win the game, absolutely. And that's why I cannot concentrate on anything else other than the game on Saturday."
Gibraltar's only other away game came in May in a 1-1 draw with Estonia, while the clash with Poland took place in front of around 1,200 fans at Estadio do Algarve in Portugal.
The prospect of playing at the 50,000-capacity Aviva Stadium will be a new challenge for Gibraltar and manager Allen Bula is eager to make sure that his players do not shrink under the spotlight.
"It is going to be a tough test again for my players, especially in a stadium that looks to be packed, but it’s about keeping talking to them, making them believe in themselves," Bula said in quotes reported by Sky Sports News.
"They've only got 30 seconds to take in the atmosphere and after 30 seconds, they've got to snap out of it."
