Fernando Santos's men ran out 4-0 victors over the Czech Republic in their most recent encounter, while their Iberian counterparts lost their grip on top spot with a 2-1 loss to Switzerland.
Match preview
No Joao Cancelo? No problem for Portugal. The Manchester City man was forced to watch Saturday's game with the Czech Republic from the naughty step, but Diogo Dalot deputised exceptionally well with a brace in that 4-0 triumph.
Diogo Jota and Bruno Fernandes also got their names on the scoresheet as Santos's side gave themselves the best possible chance of making the Nations League finals, and with it the chance to earn their second title in the tournament in three years.
With 10 points accrued from a possible 15, the 2018-19 champions have usurped Spain to first place in the table with one game to go, and a draw would be enough for the Selecao to advance to the four-team finals, but three points will still be of paramount importance.
The Czech Republic's failure to make the net ripple on Saturday represented Portugal's third clean sheet from their last four Nations League outings, and the Selecao have only conceded two goals in the entire tournament - the best defensive record from any of the 16 teams in League A.
Santos's side also welcome Spain to their headquarters having won each of their last four home matches in all competitions, and they have only ever failed to win two of their nine Nations League games in front of their own supporters so far.
Victory for Spain over Switzerland would have kept them in the driving seat no matter what on matchday five, but the fate of Luis Enrique's side is now out of their own hands after a surprise 2-1 defeat to the Rossocrociati at the weekend.
Jordi Alba had levelled for La Roja after Manuel Akanji's 21st-minute opener, but Breel Embolo struck not long after the former had restored parity for Spain to condemn the hosts to their first home defeat in nearly four years.
Enrique - who controversially went without a recognised striker as Alvaro Morata warmed the bench - pulled no punches after the game, claiming that his crop made more errors across the 90 minutes than in any previous Roja match he had taken charge of.
Now forced to adopt a gung-ho approach in their bid to make the Nations League finals, only a win will do for last year's runners-up in Braga, but Alba's equaliser on Saturday did at least mark a 19th consecutive game in which Spain had found the back of the net.
Enrique's outfit were forced to settle for a 1-1 draw against Portugal on matchday one, with Ricardo Horta coming up with a late response to Morata's opener, and that result marked the fourth stalemate in a row between the two Iberian nations - such a scoreline would benefit the Selecao more than the visitors here.
Portugal Nations League form:
D
W
W
L
W
Portugal form (all competitions):
W
D
W
W
L
W
Spain Nations League form:
D
D
W
W
L
Spain form (all competitions):
W
D
D
W
W
L
Team News
Portugal full-back Cancelo will return from suspension for the visit of Spain, but Dalot certainly does not deserve to be dropped after his two-goal showing on matchday five, so the Man City man could revert to his left-back role here.
Mario Rui and Nuno Mendes would be consigned to the bench if that is the case, although the former provided the assist for Fernandes's goal on Saturday, and Santos also witnessed blood seeping down Cristiano Ronaldo's face after a clash with Tomas Vaclik.
The Manchester United veteran was able to see out the match, but Jota is more than capable of deputising in the number nine spot after coming off the bench to round off the scoring against the Czechs.
As for Spain, Dani Carvajal and Diego Llorente both failed to make the squad for the defeat to Switzerland, with Enrique supposedly leaving both defenders behind due to the fact that they would have missed the clash with Portugal if they were booked at the weekend.
A well-rested Carvajal can therefore expect to come in for Cesar Azpilicueta on the right-hand side, and Enrique certainly ought to consider a recall for Morata after Asensio was taken off just after the hour mark on Saturday.
The Barcelona-themed midfield of Gavi, Pedri and Sergio Busquets could also be broken up given the quick turnaround, as Rodri and Koke return to a refreshed Roja team.
Portugal possible starting lineup:
Costa; Dalot, Dias, Pereira, Cancelo; Neves, Palhinha, Fernandes; B. Silva, Ronaldo, Leao
Spain possible starting lineup:
Simon; Carvajal, Garcia, Torres, Alba; Koke, Rodri, Pedri; Sarabia, Morata, Torres
We say: Portugal 2-1 Spain
Spain's frontline did not cut the mustard against Switzerland, but Enrique's side always seem to have a knack for scoring goals, and they will be forced to go all out on that front if they are to stand a chance of progressing.