it is part of the entertainment industry's ongoing love-hate relationship with ai. many studios want to make use of the technology but are concerned that their creations could be stolen.
mid journey's image generator makes images from typed requests or prompts.
in the lawsuit filed in federal district court in los angeles, the studios gave examples of midjourney-generated images that included disney characters such as star wars' yoda and marvel's spiderman, the incredible hulk and iron man.
disney's chief legal officer horacio gutierrez said the firm was "optimistic" about how ai "can be used responsibly as a tool to further human creativity".
"but piracy is piracy, and the fact that it's done by an ai company does not make it any less infringing," he said.
in the complaint, disney and universal said midjourney made $300m (£221m) last year alone and is planning a "soon-to-be-released video service".
syracuse university law professor shubha ghosh said: "a lot of the images that midjourney produces just seem to be copies of copyright characters that might be in new locations or with a new background."
"it doesn't seem like they're being transformed in a creative or imaginative way."
he added that there is a recognition in copyright law that creativity can build on other works as long as it adds something new.
randy mccarthy, head of the ip law group at us law firm hall estill said: "no litigation is ever a slam dunk, and that is true for disney and universal in this case."
"there are several issues such as terms of service provisions by midjourney, and basic fair use analysis, that will need to be sorted out by the court before we can determine the likely outcome," he added.
midjourney did not immediately respond to a bbc request for comment.
on its website, the san francisco-based startup says it has a "small self-funded team" with less than a dozen full-time staffers.
it refers to itself as "an independent research lab."
the firm is run by david holz, who previously founded a hardware sensor firm called leap motion.
midjourney lists former github chief executive nat friedman and philip rosedale, founder of second life, among its advisors.
hollywood sees both potential upsides and downsides to ai.
it was only two years ago that actors and writers shut down the entertainment industry hub with strikes demanding protections against new technology.
but now ai is being used more in tv, films and video games.
two movies competing at the oscars used ai to alter voices: emilia perez and the brutalist.
the technology has also been used to de-age actors like tom hanks and harrison ford.