Alexander Pinczowski of New York was killed along with his sister Sascha Pinczowski in the Brussels terror attack in March last year.
His wife Anne Cameron Cain accused Twitter of aiding and abetting ISIS by acting as the terror group's communication, recruiting and marketing arm, New York Post reported.
Cain has filed suit in federal court in Manhattan.
The new lawsuit calls the social media giant a "tool and a weapon of terrorism", the post reported.
Cain was joined in the Manhattan federal lawsuit by family members of Nohemi Gonzalez, a California woman who died in the 2015 Paris attack in which 130 people were killed.
"The conduct of Twitter was a direct, foreseeable and proximate cause of the wrongful deaths of Nohemi Gonzalez, Alexander Pinczowski," the lawsuit said.
The families of Pinczowski and Gonzalez are seeking unspecified damages to be determined at trial.
This is not the first time Twitter has been blamed for its use by terrorists. Legal action was initiated against Twitter, Facebook and Google following the Orlando nightclub shooting in June last year.
All of the lawsuits accuse Silicon Valley companies of not doing enough to stop terrorists from using its service to expand their reach.
In August last year, Twitter indicated that it had closed some 360,000 accounts for promoting terrorism.
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