Yes, one litter of puppies can have multiple fathers, so long as the mother was bred with multiple partners during a time when she had viable eggs. Since multiple eggs are released by the mother, sperm from different fathers could fertilize each egg.
It is even possible in humans for twins to have two different fathers. This rare event is called superfecundation and is possible if the female ovulates twice (or more) and the female has sexual relations with two different men within a couple of days of ovulation.
Superfetation is when a woman gets pregnant after already being pregnant. This results in fetuses of different ages, who may or may not share the same father.