French favourites Julie Coin and Mathieu Montcourt enjoyed safe passages through to the second round, but compatriot Mathilde Johansson was unable to use the crowd's advantage, falling to Vitalia Diatchenko 2–6, 6–2, 10–8.
Former French Open champion Gastón Gaudio made a disappointing start to his grand slam comeback, losing in straight sets to Radek Štěpánek 6–3, 6–4, 6–1.
However, more good news for American fans followed when Fed Cup heroine Alexa Glatch stunned Flavia Pennetta 6–1, 6–1, to advance to the second round.
Kateryna Bondarenko upset Patty Schnyder, and 13th seed Marion Bartoli recovered in an all-French affair to beat Pauline Parmentier 3–6, 6–1, 6–3.
Two more French wild cards fell as Uzbekistan's Akgul Amanmuradova defeated Irena Pavlovic and Australian Jarmila Groth beat Kinnie Laisné.
Another of the day's upsets came at the hands Colombia's Mariana Duque Mariño, who beat former Top 5 player and 26th seeded Russia's Anna Chakvetadze, winning 3–6, 6–4, 6–4.
The United States' hopes of having a French Open champion took a big blow as James Blake and Mardy Fish both lost to Argentinian opponents.
Fish lost to Máximo González 6–3, 1–6, 6–4, 7–6(4), and Blake, the highest seed to fall on Day 3, was beaten convincingly by Argentinian qualifier Leonardo Mayer 7–6(6), 7–5, 6–2.
There were victories for 10th seeded Dane Caroline Wozniacki, former finalist Svetlana Kuznetsova, Olympic gold medallist Elena Dementieva and World No.
World number 1 Dinara Safina was first up on Court Suzanne Lenglen, and made light work of Vitalia Diatchenko, winning 6–1, 6–1.
French tennis fans had reason to cheer as number 15 seed Alizé Cornet resumed her match from day 3, and beat Maret Ani 6–4, 7–5 to advance to the second round.
Unseeded Maria Sharapova faced number 11 seed Nadia Petrova in her first match against a top 20 player since her 10-month absence from the tour due to injury.
Defending champion Rafael Nadal made history by becoming the first person to win 30 consecutive matches at Roland Garros by beating Teymuraz Gabashvili.
1 team Liezel Huber and Cara Black, Květa Peschke and Lisa Raymond, defending champions Anabel Medina Garrigues and Virginia Ruano Pascual, and Samantha Stosur and Rennae Stubbs were amongst the first to advance to the second round of the women's doubles.
On the men's side, defending champions Pablo Cuevas and Luis Horna, Lukáš Dlouhý and Leander Paes, Bruno Soares and Kevin Ullyett, and Mariusz Fyrstenberg and Marcin Matkowski all advanced to the second round.
Schedule of play Day 5 got off to a slow start from a French perspective, as number 13 seed and home favourite Marion Bartoli succumbed to Tathiana Garbin of Italy in straight sets on Court Philippe Chatrier.
However, they did have some consolation, as Virginie Razzano navigated her way past Anabel Medina Garrigues, and booked a place in the third round to play Bartoli conqueror Garbin.
Venus Williams was made to work very hard to make the third round, as she went the distance with Lucie Šafářová, narrowly winning the final set 7–5.
There was only one shocking exit from the men's doubles on the day, as Jeff Coetzee and Jordan Kerr were knocked out by the hands of Jaroslav Levinský and Igor Zelenay.
Bob and Mike Bryan, Daniel Nestor and Nenad Zimonjić, Rik de Voest and Ashley Fisher, and Łukasz Kubot and Oliver Marach all secured places in the second round of the doubles.
There no upsets in the women's doubles, as all seeds made safe progress through to the second round including Serena and Venus Williams, and Květa Peschke and Lisa Raymond.
Cara Black and Leander Paes, Lisa Raymond and Marcin Matkowski, Nadia Petrova and Max Mirnyi, and Elena Vesnina and Daniel Nestor all advanced to the second round.
Rafael Nadal, the four-time defending champion and World Number 1, looked in stellar form as he continued his undefeated run against Lleyton Hewitt, a former holder of the top ranking, 6–1, 6–3, 6–1.
Former world number 1 Maria Sharapova continued her miraculous run by defeating Li Na 6–4, 0–6, 6–4 to reach the quarterfinals, after returning from nine months away from the game.