And fellow heroes of the 'Miracle at Medinah' also made an early exit from the European Tour's flagship £4million event, with Rory McIlroy (75), Graeme McDowell (75), Ian Poulter (76) and Paul Lawrie (72) all joining Donald in missing the halfway cut.
Donald went into the second round insisting a hat-trick was not out of the question despite struggling to an opening 78, but the world number six could only manage a 72 on another cold, wet day to finish six over par.
That was 12 shots behind Molinari, who birdied the 18th to complete a 68 and lead by one from England's Mark Foster, Scotland's Marc Warren, Spain's Alejandro Canizares and South African George Coetzee.
"I think the last couple of years it's been very warm and the ball's travelled a lot," Donald said. "I'm hitting a lot longer irons into some of these holes this year because of the weather, but the bottom line is I didn't play very well. Nine over through 21 holes is pretty terrible golf really.
"I don't think it's a million miles away though. Sometimes these things are a lot closer than you think. I've got some good time coming up with my coach back home and we'll work on it and get ready for next week and the US Open.
"I'm not too worried. It's disappointing to miss a cut, especially here at Wentworth. But I'll be back. I think failure is a much bigger motivator for me than success."
Molinari, whose halved match with Tiger Woods at Medinah sealed Europe's remarkable victory in Chicago, began his second round with a bogey and also dropped shots at the 10th and 16th, but also carded seven birdies on a course where he finished seventh last year.
The 30-year-old, who was born in Turin but is now based in London, said: "I'm really happy with yesterday [Thursday] and today [Friday]. The conditions were quite tough but I hit the ball well and the putter is working well so I am really happy at the moment.
"Six under is a good score given the conditions - I live over here now so I am used to it - and I'm in a great position going into the weekend, but it's a really tough course so anyone six or seven behind is still in it."
English rookie Eddie Pepperell birdied his last four holes to card a 69 to lie just two off the lead on four under with Italy's Matteo Manassero, with Lee Westwood and Ernie Els - playing alongside Molinari - another shot back after rounds of 71 and 69 respectively.
Westwood's chipping and putting has long been his Achilles heel, but the 40-year-old said: "My short game's been red hot. I played some nice pitches and walking off the back of the 12th green, where I pitched to about four feet, Ernie said 'Bit of a short-game wizard now'.
"I also got up and down on 18 after hitting it in the hazard and those sorts of things can make a big difference to your week and going into the weekend with momentum. If my long game starts to come together, I could be dangerous."
Sergio Garcia, embroiled in a race row since his "fried chicken" comment about Tiger Woods at the European Tour's awards dinner on Tuesday, was just five shots off the lead after a 71, with Nicolas Colsaerts a shot better off after a 70.
McIlroy had just one par, three birdies and five bogeys in a front nine of 37, but somehow scrambled seven pars in succession on the back nine despite a string of wayward drives before a double-bogey seven on the 17th ended his chances.
The world number two, again using a Scotty Cameron putter rather than the Nike club he is contracted to under his multi-million pound deal, said: "I'm definitely looking forward to getting back into some golf where I'm not playing in four layers.
"But it's the same for everyone out there and that's not really an excuse. I just didn't play well. When I play in these conditions, I sort of try and do too much with the ball sometimes, I start to hit some bad shots and those bad shots obviously cost me."
Poulter managed one birdie in a second successive 76 and has now missed the cut in his last three strokeplay events as well as losing both group matches in the Volvo World Match Play Championship last week.
"The fire's not there right now," Poulter admitted. "I've got to re-engage or throw some wood in the oven and see what happens I guess, see if we can get it to ignite again.
"Playing like that really p***** you off, so I need to get a grip and the funny thing is, I can't work any harder. So do you go 'Right, sod it all?' No, you just keep going full steam ahead. There's no panic for me."
Making his 600th European Tour appearance, Spain's Miguel Angel Jimenez birdied three of his last four holes for a 69 to finish one over.