However, there was joy for Ayermic Laporte and Rodrigo as Spain sealed their place at the 2022 Qatar showpiece with a 1-0 win over Sweden in Seville.
Bernardo, Dias and Cancelo all started in Lisbon as Portugal locked horns with Serbia in what was an action-packed Group A decider.
For Portugal, the equation was simple: avoid defeat and they would top the group above Serbia and seal their World Cup finals ticket.
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And the 2016 European champions got off to the best possible start when Renato Sanches fired them into a third minute lead from Bernardo’s neat assist.
However, a 33rd minute equaliser from Dusan Tadic set the nerves jangling in the Portuguese capital with Serbia also having a first half strike disallowed for offside.
Bernardo was substituted just past the hour mark but with the clock winding down it looked as through Portugal would hold on for the draw and so seal their World Cup finals berth.
But in injury time, Fulham striker Aleksandar Mitrovic rose to head home a dramatic winner to stun the Stadium of Light and propel Serbia into top spot, so forcing Portugal to go to a play-off.
Laporte meanwhile played the full 90 minutes in Seville to help Spain secure top spot in Group B with a narrow 1-0 win against Sweden.
It was another top of the table decider with Spain kicking off knowing a point would be enough with the Swedes requiring all three points to pip Luis Enrique’s men.
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It proved a tight, absorbing encounter with Sweden coming close on a number of occasions before the game was finally settled via Spanish substitute Alvaro Morata’s 85th minute strike.
Laporte played the full 90 minutes while City colleague Rodrigo was called on as a 60th minute substitute.
Elsewhere, Ilkay Gundogan bagged a brace of goals as Germany ended a dominant World Cup 2022 qualifying campaign in emphatic fashion via a 4-1 victory away to Armenia.
The four times world champions had long since sealed their ticket to next year’s showpiece in Qatar.
But in their final Group J assignment, City midfielder Gundogan and his colleagues rounded off proceedings in thoroughly professional fashion away in Yerevan.
Deployed at the base of the German engine room, Gundogan produced another assured display helping Hansi Flick’s side control possession and dictate play.
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Germany took early control of proceedings with Chelsea’s Kai Havertz firing them into a 13th minute lead.
And Gundogan then took centre stage as Germany doubled their advantage in first half injury time.
After Florian Neuhaus had been fouled in the box, a penalty was awarded after a lengthy VAR check.
Gundogan made no mistake from the spot, despatching a crisp shot into the bottom right-hand corner of the net.
Five minutes after the restart Gundogan struck again to further extend Germany’s advantage.
The City star despatched a fine shot from outside the box after latching onto Thomas Muller’s assist.
Former Arsenal and Manchester United midfielder Henrikh Mkhitaryan narrowed the deficit after Armenia were awarded a 59th minute penalty of their own following a foul by Florian Neuhaus.
But after Gundogan had been replaced on the hour, Germany then restored their three-goal cushion.
Jonas Hofmann’s snap-shot set the seal on a dominant win to leave Germany with 27 points from 10 games and already planning ahead for the Qatar tournament.
