Stones were thrown at two actors on their way to a performance at a theatre in what was described as a “cowardly homophobic hate crime”.
The two women were starring in Rotterdam, which tells the story of a young gay woman, at the NST Campus theatre in Southampton.
The theatre company said one of the actors was slightly injured.
The attack led to the final performances of the acclaimed play being cancelled on Saturday.
Hampshire police said it had received a report of homophobic abuse.
The Olivier-award winning play by Jon Brittain had been due for its final performances of a three-day run.
A statement from the show’s production company, Hartshorn-Hook, said the two actors were left “hugely shaken from this cowardly, homophobic hate crime”.
It said after verbally assaulting the pair, the assailants threw stones towards them from a car window on Hill Lane.
One of the women was slightly injured when she was struck on the face by a stone.
Announcing the cancellation of Saturday’s two performances, a statement said: “We are devastated that this kind of behaviour is still so prevalent, a fact which reinforces the importance of this play’s message.
“We are doing all we can to support the team and thank our audiences and colleagues for their support.”
NST director Sam Hodges tweeted: “I am extremely sad that this sort of appalling behaviour is still happening anywhere, let alone in a city where we have worked so hard to promote a culture of tolerance, inclusivity and civic pride.”
A Hampshire police spokeswoman said: “We have received a report from a third party relating to an incident which happened on Hill Lane, Southampton.
“It has been reported that homophobic abuse was shouted at two women, and stones thrown at them, by a the occupants of a passing car.
“The two women involved do not wish to pursue any police action.”
The incident comes a few days after it was revealed that two women were left covered in blood following a homophobic attack on a night bus in London.