Who is the main villain in the first two-part episode of 'Friendship Is Magic'?

The main villain in the first two-part episode of 'Friendship Is Magic' is Nightmare Moon.

Nightmare Moon appears in the 2010 series premiere, “Friendship Is Magic,” after returning from a thousand-year banishment to the moon. She threatens Equestria with eternal night during the Summer Sun Celebration, replacing Princess Celestia as the story’s central danger.

Twilight Sparkle and five new friends travel through the Everfree Forest to find the Elements of Harmony. Their friendship awakens the sixth element, Magic, allowing the group to defeat Nightmare Moon. The transformation reveals that Nightmare Moon is actually Princess Luna, Celestia’s younger sister.

The other options belong to later or separate storylines: Discord is a major villain introduced later, Queen Chrysalis leads the changelings, and Tirek becomes the principal antagonist of the fourth-season finale. Nightmare Moon is therefore the specific canonical answer for the premiere.