At least one fan could be heard voicing displeasure in Sky Sports' coverage as players from Dallas FC and Nashville knelt in protest against racial injustice.
Speaking to reporters after the match, Dallas defender Reggie Cannon said: "I think it was disgusting. I think it was absolutely disgusting.
"You got fans booing you for people taking a stand for what they believe in. Millions of other people support this cause and we discussed with every other team and the league what we're going to do and we've got fans booing us in our own stadium. How disgraceful is that? Honestly, for lack of a better word, it p****d me off.
"You can't even have support from your own fans in your own stadium. It's baffling to me."
Wednesday's contest was the first MLS match with fans since the league was suspended in March amid the coronavirus pandemic.
Cannon said Dallas had informed both Nashville and the league that the players would kneel.
He had, he said, asked for the national anthem to not be played. But on Saturday MLS Commisoner Don Garber said the anthem would be played when fans were in attendance.
In June, MLS said in a statement that it supported players' right to peacefully protest during national anthems before games, saying: "Major League Soccer stands by the ideals of freedom of speech and the right to peaceful protest that are the hallmarks of the United States and Canada."
Cannon, 22, added after the 1-0 loss, played before nearly 3,000 fans: "(Dallas player) Ryan Hollingshead, the first thing he said to me after we got up from the knee, he said 'I'm sorry.'
"I'm sorry for our fans because we had someone chanting 'USA' when they don't understand what keeling means, they don't understand why we're kneeling, they can't see the reason, they just think we're the ignorant ones and it's incredibly frustrating. And I'm sorry to have this tone but you have to call it for what it is.
"I even knew when we decided to kneel, I knew it was going to happen. That should tell you something, I knew we were going to have some negative pushback from having a unified response over what's going on. That's the problem, that's a problem."
CNN has contacted MLS but has yet to receive a response.
In July, members of Black Players for Change (BPC), an organization which has pledged to address racial inequalities in MLS and the sport, each raised a gloved fist before the league's first match following its resumption and silently knelt for eight minutes and 46 seconds, a reference to the killing of George Floyd in May which sparked worldwide protests.
The Black players were dressed in black shirts with slogans that read "Silence is Violence," "Black All The Time," and "Black and Proud." Their black face masks said "Black Lives Matter."
The pre-match demonstration has been featured in a short documentary, which runs for eight minutes and 46 seconds, about the foundation of BPC.
Eight minutes and 46 seconds represents the duration prosecutors initially reported Minneapolis police officer Derek Chauvin knelt on Floyd's neck.
Prosecutors have
