Isner, the world number 12, battled hard past Australian Matosevic to post a 7-6 (10-8) 6-4 victory in one hour 41 minutes at the hard-court event.
The American sent down 17 aces to Matosevic's seven, however was forced to save two set points in the first set tie-break before continuing to the last four.
Isner was broken in the first game of the contest at the ATP 250 event, but he broke back to 15 in the fourth game as the momentum swung.
Both players held a break point each in games five and six, but there would be no breaks for the remainder of the set - with Isner blowing a set point on the Matosevic serve in the 12th game.
Matosevic opened up a 3-0 lead in the tie-break, and was first to a set point as he led 6-5.
Isner saved it and denied Matosevic on another set point, and the American lost just five points on serve in the second set as he broke once to canter to victory.
Fourth seed Vasek Pospisil was upset by unseeded Israeli Dudi Sela in their quarter-final.
Sela posted a 7-5 1-6 6-2 victory in one hour 42 minutes, despite trailing early in the deciding set.
Pospisil had all the momentum, as he led 2-0 in the third, and held deuce on the Sela serve.
But the diminutive Sela would lose just four points there onwards, as he booked a semi-final date with Benjamin Becker of Germany.
Becker downed lucky loser Thiemo de Bakker of the Netherlands 6-4 6-2.
Isner's last-four opponent will be fellow American Jack Sock, who continued his impressive 2014 with a come-from-behind 7-6 (8-6) 6-2 win over Slovakian Lukas Lacko.
Lacko served for the first set at 5-3 up, having lost just four points on serve in as many games, but he was broken to 15 as Sock scraped his way into the contest.
Sock clinched an ugly tie-break - with half of the 14 points going against serve - before the rising American effected three consecutive breaks in the second set to seal the win in one hour 20 minutes.