Level 3, which carries video for the video-rental company Netflix, accused Comcast of "putting up a toll booth at the borders of its broadband Internet access network."
Comcast said it is treating Level 3 the same as its competitors.
Level 3 is "claiming it's entitled to be treated differently and trying to force Comcast to give [it] unlimited and highly imbalanced traffic and shift all the cost onto Comcast and its customers," according to a statement from Comcast.
Analysts at Stifel Nicolaus wrote in a note on Tuesday that the allegations "could further complicate [Comcast's] push to receive final regulatory approval for the NBCU deal by year-end."
Meanwhile, cable modem maker Zoom Telephonics filed a complaint at the FCC on Monday claiming that the cable company is preventing subscribers from attaching cable modems to the network, an alleged violation to net-neutrality policies.
The complaints also coincide with deliberations at the FCC over whether to strengthen its net-neutrality policies by creating formal rules to govern broadband networks. Right now, the FCC only has a net-neutrality policy statement, but Genachowski has long said he wants official rules.
He could indicate whether he will move forward with formal rules as soon as Tuesday evening.