The Giant Spiders are minor antagonists in the first arc of the My Little Pony: Friendship Is Magic IDW comic series The Return Of Queen Chrysalis.
The Giant Spiders are obviously styled after stereotypical gangsters, given their clothing. Their mandibles are reminiscent of moustaches. Each spider has a distinctive mark on its abdomen, reminiscent of a Cutie Mark. The leader of the group seems to be a very large red tarantula wearing two eyepatches.
The Spiders' signature ability is to project large amounts of sticky, steel-strong silk from a long range, which allows them to easily overpower the Mane Six. In addition, the red tarantula seems to be particularly resistant to magic, as a strong bolt from Twilight Sparkle seems to deal almost no damage.
Depiction in the comics
A group of dangerous giant spiders attack the Mane six during their travel to rescue Apple Bloom, Scootaloo and Sweetie Belle from Queen Chrysalis and her army of changelings. Their friendship damaged by a ploy from the changelings, the Mane Six cannot cooperate well enough to save themselves from the threat. Applejack, Rainbow Dash, Fluttershy and Rarity all end up encased in webbing, and Pinkie Pie runs away, leaving Twilight to fend for herself.
When Twilight starts using her magic, the smaller spiders seem to be scared and elope, leaving the red tarantula to deal with her. Twilight fires a beam at the tarantula's face, but it mostly just alloys the giant beast.
Just then, the day is saved by Pinkie Pie who, as it turns out, has notified Jim the Cave Troll, who considers the biggest spider a "teddy bear". He forcibly drags the spider away with him, saying he will name it "F'wuffy".