The home side edged the reverse fixture 3-1 in Hoffenheim in November.
Urs Fischer's side made the better start in the game, with Robert Andrich narrowly firing wide from distance in a relatively quiet first half. After the break, Marcus Ingvartsen's lovely cross from the right found Grischa Promel at the back post, with the central midfielder heading home to give the visitors a deserved lead.
Despite being on a superb run of form, Freiburg offered very little throughout the game, struggling to break down Union Berlin's stubborn defence. Fischer's side squandered several opportunities in transition to double their lead, too, with Joel Pohjanpalo particularly profligate, but they comfortably held onto their lead to leapfrog their opponents in the table.
Having only been promoted two seasons ago, finishing in a Europa League qualification position would have been almost unthinkable for Eiserne fans this season. However, with top goalscorer Max Kruse now fit again after recovering from his torn muscle, there is every chance that they could achieve exactly that. Fischer will be urging his players to take one game at a time, though, starting with a very winnable home game against Hoffenheim on Sunday.
Sebastian Hoeness's side certainly have the disadvantage of having had far less rest, having crashed out of the Europa League after a 2-0 defeat at home to Molde on Thursday evening.
In many ways the defeat summed up the unfortunate nature of Hoffenheim's season, with Die Kraichgauer having 27 shots, nine shots on target, 74% possession and 13 corners across the 90 minutes. In contrast, Molde's only two shots on target were Eirik Ulland Andersen's brace.
Perhaps Hoffenheim used up all of their goals in last weekend's 4-0 win over Werder Bremen, which in truth did flatter them a little. Ihlas Bebou opened the scoring with another fine display of composure in front of goal, before Christoph Baumgartner doubled the hosts' lead moments before the break after too easily being allowed to jink his way into the Bremen penalty area and stab the ball home.
Munas Dabbur and 18-year-old debutant Georginio Rutter added gloss to the victory, which took Hoeness's side nine points clear of the relegation zone. While their fans will understandably be distraught at exiting the club's first ever European knockout stage at the first hurdle, at least they should be able to breathe easily regarding their Bundesliga status after what has been a testing campaign.
Union Berlin Bundesliga form: LLDLDW
Hoffenheim Bundesliga form: WWLLDW
Hoffenheim form (all competitions): LLDDWL
Fischer will be delighted to have Kruse available once again, although the attacking midfielder was not used from the bench during the win over Freiburg, so may need to be eased into things before he is named in the first XI.
Liverpool loanee Taiwo Awoniyi and Sheraldo Becker are both ruled out through injury, leaving the hosts a little light in attack, with Niko Giesselmann and Anthony Ujah both remaining on the treatment table, too.
Hoffenheim's injury crisis, meanwhile, shows little sign of easing, with Ishak Belfodil, Kostas Stafylidis, Mijat Gacinovic, Stefan Posch, Robert Skov, Havard Nordtveit, Kevin Akpoguma, Dennis Geiger, Ermin Bicakcic and Benjamin Hubner all likely to be ruled out of the trip to Berlin.
Andrej Kramaric is available once again, though, and could be set to start after twice gaining important match fitness from the bench within the past week.
Union Berlin possible starting lineup:
Karius; Friedrich, Knoche, Schlotterbeck; Trimmel, Promel, Andrich, Ingvartsen, Lenz; Bulter, Pohjanpalo
Hoffenheim possible starting lineup:
Baumann; Kasim, Grillitsch, Richards; Kaderabek, Rudy, Samassekou, John; Baumgartner; Kramaric, Bebou
We say: Union Berlin 1-1 Hoffenheim
Much will hinge on whether Kruse and Kramaric start for their respective sides, with both players capable of making a huge impact with their goals and creativity.
Hoffenheim do tend to prefer playing away from home, so we can envisage them gaining a positive result on Sunday.