The Saudi Pro League side stayed on track for a record fifth title as Mitrovic put them ahead four minutes into added time and Al Hamdan doubled the advantage three minutes later.
Those goals dealt a killer blow to Sepahan's hopes after Ramin Rezaeian had given the hosts the lead in the 37th minute.
That goal was cancelled out by Malcom's close-range strike 12 minutes after the restart and Mohammad Daneshgar's sending-off in the 76th minute swung the tie in Al Hilal's favour.
The teams meet again in Riyadh next Thursday.
Rezaeian put Sepahan in front after evading Al Hilal's attempts to play a high defensive line and the Iran international controlled the ball before smashing a right-foot shot past Yassine Bounou.
But Al Hilal silenced the Naghsh-e Jahan Stadium in the 57th minute when Mitrovic's strike was kept out by Payam Niazmand, only for Malcom to slide the rebound under the goalkeeper's body.
Daneshgar's reckless tackle on Al Hamdan earned him a red card 14 minutes from time and Al Hilal took control.
Mitrovic rose above Rezaeian to head in Saud Abdulhamid's cross from the right and Al Hamdan sealed the win with a clinical finish.
Al Ittihad, meanwhile, were held to a 0-0 draw by Uzbekistan's Navbahor in Namangan as the Saudi Pro League champions fielded a side missing former France international striker Karim Benzema.
Former continental champions Ulsan Hyundai put one foot in the quarter-finals on Thursday as the South Koreans handed Japanese second division side Ventforet Kofu a 3-0 defeat at Ulsan Munsu Stadium.
Joo Min-kyu scored twice in eight minutes late in the first half to put Ulsan in control and Seol Young-woo netted his side's third 16 minutes after the interval.
The teams meet in Tokyo next week with Kofu, the first second division side to qualify for the knockout rounds of the competition, facing an uphill task to overturn the deficit.
Jeonbuk Motors won 2-0 in the first leg of their battle with fellow South Koreans Pohang Steelers on Wednesday.
Kawasaki Frontale from Japan hold a 3-2 advantage over Chinese Super League side Shandong Taishan while Yokohama F Marinos gave up a two-goal lead to draw 2-2 with Bangkok United.
In the western half of the draw, Cristiano Ronaldo scored the only goal as Al Nassr beat fellow Saudi side Al Fayha on Wednesday, when Al-Ain of the United Arab Emirates drew 0-0 with Nasaf from Uzbekistan.
