The first leg will be played at Stadion An de Alten Forsterei on Thursday, with the return leg taking place in Belgium next week.
Match preview
This is unchartered waters for both clubs, who have already made it further than ever before in any European competition in their respective histories, with Union Berlin's previous best effort coming with a second round defeat in the 2001-02 UEFA Cup.
Despite finishing second to their forthcoming opponents in Group D, head coach Urs Fischer will sense a serious opportunity to reach the last eight of the Europa League, with his side entering the tie as favourites to progress.
Having seen their Bundesliga title charge derailed by a winless streak of three matches in recent weeks, Die Eisernen are set to throw everything at this competition, as they look to qualify for the Champions League through either their league position or winning Europe's secondary trophy.
Their 3-1 win against Ajax on home territory in the previous round will undoubtedly have significantly boosted their confidence of making it far, but they will almost certainly be pushing hard for a first leg lead for them to protect in Belgium next week.
Union SG, meanwhile, have made a similarly remarkable rise in recent seasons, having only achieved their first promotion to Belgium's top flight for 48 years at the end of 2020-21 campaign.
Les Unionistes subsequently became the first team in Belgian history to finish top of the table at the end of the regular season directly after promotion, although they missed out on claiming their first top-flight title since 1935 after finishing second to Club Brugge in the end of season play-offs.
While they still retain aspirations of going one better domestically this season, head coach Karel Geraerts will be desperate to build on topping their Europa League group prior to the World Cup by progressing beyond Union Berlin at the last-16 stage.
Having already beaten them once at Stadion An de Alten Forsterei this season, Geraerts will have every confidence that his players can deliver a positive result in Germany's capital on Thursday to set themselves up nicely for the return leg at Joseph Marien Stadium next week.
Union Berlin Europa League form:
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Union Berlin form (all competitions):
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Union SG Europa League form:
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Union SG form (all competitions):
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Team News
Union Berlin will remain without Andras Schafer due to a foot injury, with the Hungary midfielder facing a race against time to be fit before the international break later this month.
Niko Giesselmann and Jerome Roussillon both missed their side's 0-0 draw against FC Koln at the weekend due to knocks, but the duo should be available for selection on Thursday, especially for a match of such magnitude.
Fischer's biggest dilemma will be whether to restore Jordan Siebatcheu to his starting XI at the expense of Kevin Behrens, with neither forward scoring in any of their last six appearances in all competitions.
Union SG, meanwhile, may travel without Dennis Eckert Ayensa due to a muscle strain, but otherwise Geraerts appears to have a fully fit squad at his disposal.
Victor Boniface is likely to replace Gustaf Nilsson in attack after only being used from the bench during their 2-1 victory against Eupen at the weekend, with the Nigeria forward scoring three goals in five starts during the group stage.
Union Berlin possible starting lineup:
Ronnow; Doekhi, Knoche, Leite; Juranovic, Thorsby, Khedira, Haberer, Roussillon; Siebatcheu, Becker
Union SG possible starting lineup:
Moris; Sykes, Burgess, Van Der Heyden; Lapoussin, Lynen, Teuma, Amani, Adingra; Vertessen, Boniface
We say: Union Berlin 1-0 Union SG
Both meetings at the group stage ended 1-0 to the away team, but we can envisage a reverse scoreline on Thursday, with Union Berlin likely to be able to keep their opponents at arms' length.
The two managers deploy very similar formations with an emphasis on remaining compact out of possession, so it may not be a match for the purists to savour. However, the home fans will be roaring their team onto unprecedented success, with Union SG potentially having to settle for keeping the tie alive heading into the second leg.