Anointed as the player to take over Williams' mantle, Muguruza has subsequently failed to reach a single final at any tournament.
Speaking about Mladenovic's performance, Muguruza added: 'She is playing good. She was a set and a break down in her second round match against the in-form Anett Kontaveit but looked to have settled back into the rhythm of her game until she came up against home-crowd hope Kristina Mladenovic.
Mladenovic often played to the fans, waving her arms to ask for more noise.
Carina Witthoeft turned up late for her third round match against second-seeded Karolina Pliskova at the French Open.
She returned to her baseline, miffed, but never channeled that frustration into her trademark power strokes.
Mladenovic defended her support, saying: "I lost a final in Stuttgart against Laura Siegemund, a German player, and I thought the crowd was extremely tough there".
About an hour after Muguruza's exit, seven-time major champion Williams lost to 30th-seeded Timea Bacsinszky 5-7, 6-2, 6-1 at Court Philippe Chatrier. But the swirl of emotions defending a Grand Slam for the first time is something else.
They aren't the only two with more than a puncher's chance.
"I understand. I just think that they were a little bit, sometimes should be a little bit more respectful".
Muguruza's defeat means Justine Henin, who won the title in 2003 and then from 2005 to 2007, remains the last woman to successfully defend her crown at Roland Garros.
"I wanted to go as far as possible, but even if I didn't, I think I'm going to feel much better now to continue the year, and everybody is going to stop bothering me asking me about this tournament". Her best French Open showing was in 2002, when she lost to Serena in the final.
Two weeks past his 21st birthday, Karen Khachanov became the youngest man to reach the fourth round in Paris since 2009, beating 21st-seeded John Isner - the last American man in the tournament - 7-6 (1), 6-3, 6-7 (5), 7-6 (3).
Meanwhile, Caroline Wozniacki also advanced to the French Open quarterfinals for the first time since 2010 after defeating Svetlana Kuznetsova 6-1, 4-6, 6-2. You give me the courage and strength to keep going in every point. Second seed Karolina Pliskova was the most notable victor, while Elina Svitolina, Petra Martic and Veronica Cepede Royg also progressed to the last 16.
The 22-year-old Ukrainian, who lives and trains in Paris, put on an untidy display but played perfectly on the key points, holding serve throughout.