Louis van Gaal named both Depay and De Jong in the first XI for their trip to Warsaw in League A Group 4, where goals from Cody Gakpo and Steven Bergwijn secured a 2-0 win for the Netherlands.
However, Van Gaal suffered no fewer than three fresh injury concerns in midweek, as midfielder Teun Koopmeiners came off with concussion after five minutes.
De Jong then had to be withdrawn at the half-time interval, and seven minutes into the second half, Depay was switched out for Vincent Janssen.
The Dutch Football Federation have now announced that both De Jong and Depay will not play in their final Nations League game against Belgium on Monday.
"Teun Koopmeiners left the training camp this afternoon after returning from Warsaw. The midfielder unfortunately collided with an opponent in the opening phase directly in the opening phase and suffered a concussion as a result," a statement read.
"The training camp has also come to an end for Memphis, who had to be substituted at the start of the second half due to an upper leg injury. Frenkie de Jong, who did not return to the field after the break in the duel with Poland, also goes home. He also has problems with his thigh."
Brian Brobbey and Ryan Gravenberch have been called up to the senior squad in Depay and De Jong's absence, as Van Gaal's men aim to secure their place in the Nations League finals.
Oranje have taken 13 points from five games to storm to the top of the rankings - three clear of second-placed Belgium - meaning that a draw would be enough for Van Gaal's side to advance to the finals.
The extent of De Jong and Depay's injuries are unclear at present, but the pair have emerged as doubts for Barcelona's first game back against Mallorca in La Liga on October 1.
La Blaugrana have remained relatively injury-free during the early stages of the season, and neither Depay nor De Jong have been regular starters in the 2022-23 campaign so far.
The latter has only started four of his side's eight matches in all competitions, while Depay has only made three appearances but was handed a start against Elche on September 17.
The 28-year-old repaid Xavi's faith in him with Barcelona's second goal in that 3-0 win and was delighted to grasp his opportunity with both hands, telling the press: "I'm happy to score.
"When opportunities come to me, I try to contribute to the team. I know what I can offer, and I show that in training. I'm happy to be given a chance today. I'm not playing much this season, but I'll keep on working in the same way.
"I need more match rhythm to give my best for the team. I know I have the ability. I'm happy to go to the national team now, then come back with the desire to win titles in this club."
Barcelona sit second in the La Liga table and could briefly go top with victory over Mallorca on October 1 before current leaders Real Madrid host Osasuna 24 hours later.