Osorio said the incident halted Mexico's momentum as they chased the game following Neymar's opening goal, and that referee Gianluca Rocchi "favoured Brazil".
The five-time World Cup winners went up a gear and dominated the rest of the game as the Mexicans ran out of steam with Willian and Neymar going close. It turned out to be pure histrionics.
Neymar scored Brazil's opener in the win and set up Roberto Firmino for their second as he continues to work his way back to full-match fitness after suffering a broken bone in his right foot playing for PSG in February.
While Layun didn't receive a caution for his action, he clearly stepped on Neymar and enabled a stoppage that halted some of Mexico's momentum.
He had been Brazil's greatest threat in the first half and he had scored the opening goal in the second, unlocking the Mexican defense with an astute backheel and then tucking in Willian's low cross.
Brazil's talisman was up to his usual tricks again in the Selecao's last-16 victory against Mexico, with Tite's side winning 2-0.
Mexico has now lost at this stage at every tournament over the same period of time, extending its wait for the "quinto partido" - or fifth game - for at least another four years.
Replays appeared to show Layun caught Neymar on his ankle but he escaped sanction. "This should be a game of men and not so much clowning around". They caused problems for Brazil, too, initially.
That is stretching the point, in truth: Mexico's early vim had long since faded by the time the handbags started and their coach surely knows better than to deflect from a performance that faded sharply.
During the win over Costa Rica in the group stage, Neymar tried to win a penalty by flopping to the ground in the area.
Minutes after the restart Brazil took the lead in exquisite fashion with Neymar starting and ending the move.
But, just as Brazil had done early on, Mexico's defense would bend but not break, defending chances from Neymar and Coutinho. "He just has to play, when it's a situation about refereeing that's [the coaches'] responsibility to talk about it". "We were maybe not very efficient and we didn't have that extra quality that they had coming to our goal".