This line is difficult to explicate precisely, but we can be fairly certain that the implication of the line is that the "two Indian ponies" are experiencing (and representing) a moment of affection.
The poem has already stated that twilight "bounds softly onto the grass," thus suggesting that the light of day is itself darkening. This could be taken as a partial explanation for the darkening eyes of the ponies. (Their eyes are darkened because they are now in twilight and because they are taking on the attributes of the gentle light.)
We might also wonder if there is an additional notion at play wherein the darkening of the eyes is the result of blood rushing to the face of the animals, like a blush (even, perhaps, an amorous blush, albeit innocently amorous).
There are overtones here of seduction. The darkening light, the darkening eyes and other details generate this allusion.
And the light breeze moves me to caress her long ear
That is delicate as the skin over a girl’s wrist.
The ponies may also be understood to be batting their eyes or partially closing their eyes in ways that also relate to seduction.
In this way the darkening eyes can be read as a implicit allusion to seduction, yet the poem's romance is rather Romantic (meaning that nature here represents a body of ideas that are almost metaphysical and the energy of love and affection can be related to an embrace of Being and a sense of connection to the natural world).