Nato left it until the final minute of the 30-minute session to set the best lap of 1m09.101s, eclipsing Maserati MSG driver Guenther by 0.139s.
Sam Bird was third-fastest for Jaguar, just two thousandths further back, posting his best time after the chequered flag fell.
For much of the session it had been McLaren driver Rene Rast in control of the timesheets, the triple DTM champion having been fastest in Friday evening's opening practice session.
Rast went to the top of the leaderboard at around the seven-minute mark on a 1m10.515s, and was first to break into the 1m09s just minutes later with a 1m09.395s.
The German driver subsequently improved to a 1m09.255s, leading a McLaren 1-2 ahead of team-mate Jake Hughes heading into the final 10 minutes, before Jakarta E-Prix winner Guenther jumped to the head of the times.
Rast's time was still enough for fourth, although his best effort from first practice, a 1m09.054s, stands as the best of the weekend so far.
Jean-Eric Vergne was fifth-fastest for DS Penske, despite suffering an off at the final corner in the latter stages of the session.
Sacha Fenestraz was sixth in the second of the factory Nissan, while McLaren's Hughes ended up seventh, meaning all four Nissan-powered cars featured in the top seven.
Completing the top 10 were Edoardo Mortara and the Porsches of points leader Pascal Wehrlein and Antonio Felix da Costa.
Wehrlein's nearest rival in the championship, Andretti driver Jake Dennis, was a lowly 19th after losing a chance to improve on his laptime late on when he encountered a dawdling Sergio Sette Camara (NIO 333) at the final corner, forcing him to take avoiding action.
Nick Cassidy, third in the points, was a low-key 20th for Envision after suffering an off of his own at the same point of the track, while another title contender, Jaguar's Mitch Evans was a low-key 21st.
Qualifying for the Portland E-Prix is scheduled to begin at 12:40pm local time, with the race beginning at 5pm.
