The USL club made headlines after eliminating in-state Columbus Crew SC in the fifth round and needed penalties to sneak past a second-straight MLS club after a scoreless first 120 minutes.
Cincinnati had a goal controversially ruled out for offside late in the second half, while goalkeeper Mitch Hildebrandt made a number of fine saves to frustrate Bastian Schweinsteiger and the MLS stars.
AndHildebrandt was the star in the shootout as well, stopping attempts from MLS leading scorer Nemanja Nikolic as well as Arturo Alvarez and Juninho for another famous FC Cincinnati victory.
The nationally televised game drew 32,287 fans to Nippert Stadium in Cincinnati, one of 12 cities currently competing for an opportunity to move to MLS.
"We have an MLS-quality team, we have an MLS-quality stadium, we have MLS-quality fans," Hildebrandt said after the win. "Everyone here, the city deserves this."
Also on Wednesday night, Kwadwo Poku's stoppage-time goal gave Miami FC a stunning upset of its own, beating Atlanta United 3-2 at Riccardo Silva Stadium, two weeks after the NASL club also eliminated Orlando City.
Stefano Pinho's 37th-minute goal cancelled outBrandon Vazquez's opener for Atlanta.Rhett Bernstein put Miami temporarily ahead in the 52nd minute beforeJulian Gressel evened the score from the penalty spot 15 minutes from time.
But Poku delivered three minutes into added time, turning homeVincenzo Rennella from close range to send Miami to the quarterfinals next month, when the club will host FC Cincinnati.
The other lower-division side still in the tournament did not fare as well, as the Sacramento Republic fell 2-0 to the LA Galaxy.
The MLS giants broke the deadlock two minutes after halftime from Ariel Lassiter's free kick, before Bradford Jamieson added a second goal two minutes after that.
The Galaxy will next travel to meet the San Jose Earthquakes, who fought off the 10-man Seattle Sounders 2-1.
San Jose's Shea Salinas needed just six minutes to open the scoring, and things got worse for Seattle as Francisco Narbon was sent off on 35 minutes.
But though the Sounders were able to equalize through Aaron Kovar early in the second half, Danny Hoesen showed off remarkable control in the box before netting the 84th-minute winner.
Meanwhile, Sporting Kansas City advanced to the next round with a 2-0 defeat of Houston Dynamo atBBVA Compass Stadium.
Ike Opara opened the scoring in the 61st minute, heading in Kevin Ellis' header from a corner kick. Gerso Fernandes then sealed SKC's passage in second-half stoppage time, turning homeLatif Blessing's pass from a counter-attack.
Sporting KC will next host defending champions FC Dallas, which beat the Colorado Rapids 3-1 on Tuesday night.
The New England Revolution edged D.C. United 2-1 at Harvard University's Jordan Field.
Jose Ortiz put the visitors in front after seven minutes, but goals on either side of half-time fromDiego Fagundez and Brian Wright put the Revs in the lead for good.
New England will host the New York Red Bulls, whobeat the Philadelphia Union 5-3 on penalties after a 1-1 game.
After Sacha Kljestan gave New York the lead before halftime, Roland Albert equalized with just four minutes to play in the second half.
In the shootout, Red Bulls keeper Ryan Meara stopped Fafa Picault's penalty while all New York shooters converted.
FC Cincinnati (3) 0-0 (1) Chicago Fire: Fire stunned
FC Cincinnati stunned the Chicago Fire in the Round of 16 in the U.S. Open Cup, with goalie Mitch Hildebrandt playing hero.
Miami FC 3-2 Atlanta United: Miami shock win at the death
Miami scored deep into stoppage time to shock MLS giants Atlanta United in the U.S. Open Cup.
Galaxy 2-0 Sacramento Republic: L.A. advance
The Galaxy netted two fine second-half goals to down Sacramento Republic and move on to the U.S. Open Cup quarterfinals.
San Jose 2-1 Seattle: Hoesen plays hero
Danny Hoesen scored an 84th-minute winner to propel San Jose past four-time winners Seattle Sounders in the U.S. Open Cup.
Houston 0-2 Sporting KC: SKC move on
Second-half goals from Ike Opara and Gerso Fernandes lifted Sporting KC past Houston into the U.S. Open Cup quarterfinals.
Revolution 2-1 DC United: Wright lifts Revs
Two goals in a span of four minutes would secure a narrow 2-1 win for the Revolution over DC United in the U.S. Open Cup.
New York (5) 1-1 (3) Philadelphia: Red Bulls get revenge
The New York Red Bulls avenged two straight seasons of US Open Cup defeats to the Union with a penalty shootout win.