The message, which appeared on a militant Web site that has carried al Qaeda statements in the past and bore the logo of the extremist group's media wing al-Sahab, showed a still image of bin Laden aiming with an assault rifle.
"The response will be what you see and not what you hear and let our mothers bereave us if we do not make victorious our messenger of God," said a voice believed to be bin Laden's, without specifying what action would be taken.
The five-minute message, bin Laden's first this year, made no mention of the fifth anniversary Wednesday of the U.S.-led invasion in Iraq.
It came as the Muslim world marks the Prophet Muhammad's birthday Thursday and amid the reigniting of a two-year-old controversy over some Danish cartoons deemed by Muslims to be insulting. Islamic law generally opposes any depiction of the prophet, even favorable, for fear it could lead to idolatry.
On Feb. 13, Danish newspapers republished a cartoon showing Muhammad wearing a bomb-shaped turban to show their commitment to freedom of speech after police said they had uncovered a plot to kill the artist.
Danish intelligence service said the reprinting of the cartoon had brought "negative attention" to Denmark and may have increased the risk to Danes at home and abroad.
The original 12 cartoons first published in a Danish newspaper triggered major protests in Muslim countries in 2006. There have been renewed protests in the last month.
Ben Venzke, the head of IntelCenter, a U.S. group that monitors militant messages, called Wednesday's message a "clear threat against EU member countries and an indicator of a possible upcoming significant attack."
In the message, Bin Laden described the cartoons as taking place in the framework of a "new Crusade" against Islam, in which he said the Pope has played a "large and lengthy role."
"You went overboard in your unbelief and freed yourselves of the etiquettes of dispute and fighting and went to the extent of publishing these insulting drawings," he said, according to a transcript released by the SITE Institute, another U.S. group that monitors terror messages. "This is the greater and more serious tragedy, and reckoning for it will be more severe."
Adam Raisman, senior analyst at the SITE Institute, said that the message's release coincides with an increased buzz in online jihadi forums calling for revenge against Europe over the cartoons.
But Raisman noted that bin Laden's message did not specifically mention the republishing of the cartoons, only the publishing, and it did not give any other time landmarks to prove it had been recorded since then.
Raisman also noted bin Laden's silence on Wednesday's fifth anniversary of the U.S.-led invasion of Iraq.
"The tape doesn't give any specific evidence that would allow us to determine when it was recorded," Raisman said.
In the message addressed to "the intelligent ones in the European Union," bin Laden also criticized the "aggressive policies" of President Bush.
"How it saddens us that you target our villages with your bombing: those modest mud villages which have collapsed onto our women and children. You do that intentionally, and I am witness to that," he said, according to SITE. "All of this (you do) without right and in conformity with your oppressive ally who along with his aggressive policies is about to depart the White House."
On Wednesday, Bush praised Sunni tribal leaders for rising up against al Qaeda in Iraq and said that has led to similar uprising across the country. All that, combined with a strategic influx of U.S. troops last year, has "opened the door to a major victory in the broader war on terror," Bush said.
"Iraq was supposed to be the place where al Qaeda rallied Arab masses to drive America out," Bush said. "Instead, Iraq has become the place where Arabs joined with Americans to drive al Qaeda out. In Iraq, we are witnessing the first large-scale Arab uprising against Osama bin Laden, his grim ideology."
In Wednesday's message, bin Laden also attacked his long-time nemesis, King Abdullah of Saudi Arabia, whom he described as the "crownless king in Riyadh" and said he could have ended the entire dispute over the cartoons if he had wanted because of his influence with European governments.
Bin Laden, who hails from a powerful Saudi family, was stripped of his citizenship in 1994 after criticizing Saudi Arabia for allowing U.S. troops on its soil.
Wednesday's message, which featured English subtitles, follows up an hour-long, audio missive from Dec. 29 in which he warned Iraq's Sunni Arabs against fighting Al Qaeda in Iraq and vowed new attacks on Israel.