Nastyukhin made his MMA debut on April 26, 2010, at Siberian League: Siberia vs. Ural. He lost via rear-naked choke to Magomed Guseinov. Nastyukhin was only 18-years-old at the time of his debut, and after the loss he took nearly three years away from active competition to improve his skill set. On April 8, 2013, Nastyukhin returned to action and began a seven-fight win streak that would lead to his signing to ONE Championship against Dmitry Ermolaev at Siberian League: Siberia vs. Kazakhstan. He won via triangle choke in the first round.
ONE Championship On December 5, 2014, Nastyukhin debuted in ONE Championship against
Eduard Folayang. He made an instant impact with a first-round knockout by way of a flying knee and soccer kicks in the first round. At ONE: Defending Honor on November 11, 2016, Nastyukhin squared off against
Kotetsu Boku. During the first round, Nastyukhin broke his leg. Although he was able to finish the round, a ringside physician called a stop to the bout. Nastyukhin would not return until August 2017. In the opening round of the ONE Championship Lightweight World Grand Prix on March 31, 2019, Nastyukhin was matched up with the debuting
Eddie Alvarez. Alvarez, a former
Bellator and
UFC lightweight world champion, was seeking another major championship. Nastyukhin stunned the world with a first-round TKO over Alvarez. However, due to injury, Nastyukhin would not be able to advance to the next round and compete in the semi-finals. On November 6, 2020, Nastyukhin returned to action and faced #3-ranked lightweight Pieter Buist at
ONE Championship: Inside the Matrix 2 in a ONE lightweight title eliminator. He won the fight by unanimous decision and secured a title shot against
ONE Lightweight World Champion Christian Lee. Nastyukhin's title shot materialized against
Christian Lee at
ONE on TNT 2 on April 14, 2021. He lost the fight via TKO in the first round. Nastyukhin faced Saygid Guseyn Arslanaliev in a rematch at
ONE: Winter Warriors on December 3, 2021. He lost the bout via TKO in the third round. The contest with Arslanaliev was later named 2021 Fight of the Year by ONE Championship. Nastyukhin faced Halil Amir at
ONE on Prime Video 2 on September 30, 2022. He lost the bout via TKO stoppage at the beginning of the second round. Nastyukhin faced
Zhang Lipeng on October 6, 2023, at
ONE Fight Night 15. He lost the fight via technical knockout in the first round. Nastyukhin was scheduled to face Aaron Cañarte on January 11, 2025, at
ONE Fight Night 27.{{Cite web |date=2024-11-22 |title=Aaron Cañarte to Face Timofey Nastyukhin at ONE Fight Night 27 ==Championships and accomplishments==