Norwegian duo Heidi Weng and Therese Johaug were second and third, and defending champion Ebba Andersson came fourth after falling towards the end of the final lap.
It means Sweden have completed a "golden sweep" at Trondheim 2025, winning all six gold medals in women's cross-country events at the championships.
For 25-year-old Karlsson, it was a first individual gold medal at the World Championships, adding to her relay victories in 2019 and this week.
"It's unbelievable. I was very tired on the second lap, so I thought this would be a really tough day, but then I got some energy and I had such good skis," an emotional Karlsson said.
"Our wax team needs to have this gold medal, I think, so I'm super happy. It means a lot. It was the hardest conditions you can ever imagine."
The women's freestyle mass start, which was held over 30km until the previous edition of the world championships, played out in challenging conditions, with competitors forced to battle their way through deep and loose snow.
Andersson, Johaug, Weng and Karlsson began to pull away after 10km and increased their lead on the chasing pack, with the lead continuously changing hands between the four breakaway skiers.
Johaug, who has won the event four times, twice made attempts to drop the three other contenders, but was foiled by the Swedish duo's persistence and tactical defending.
On a frustrating afternoon for Johaug, the veteran had a tense exchange with Karlsson midway through the race, shouting at the Swede for treading on her skis.
"I went for gold today, I did it. The way the race developed, and the conditions were on the course, I was not able to use my strongest weapon, which is my capacity," Johaug told broadcaster TV2.
"I was pretty fed up with Frida stepping on my poles and skis many times. It's so unnecessary when you're a small group travelling together, when you can show some respect for the person in front."
Andersson, bidding for her fourth gold medal at the championships, made a rare error as the quartet picked up the pace heading into the final kilometre, going down after bumping skis with compatriot Karlsson.
In the confusion, Johaug moved into the lead, but she could not find enough pace to hold off Karlsson and Weng who passed her with ease.
Johaug, a four-times Olympic champion in cross-country skiing, retired from the sport shortly after winning three gold medals at the Beijing Winter Games in 2022 before reversing that decision in August last year.
The 36-year-old was beaten to the women's 20km skiathlon gold by Andersson in a thrilling finish last week, before once again finishing second behind the Swede in the 10km classic on Tuesday.
She also won a relay silver on Friday.