Mr Mattis said he agreed with the UK and Dutch assessment Moscow was allegedly responsible for the attempted hacking of the Organisation for the Prohibition of Chemical Weapons (OPCW), located in The Hague.
"I've seen enough of the evidence to say the Dutch and the British are 100 per cent accurate in who they've attributed this to," he said, adding: "We are ready today to provide cyber-support to our allies. That is now."
Mr Mattis was delivering remarks after a meeting of the North Atlantic Treaty Organisation (Nato).
Mr Mattis was echoing statements made by Dutch Defence Minister Ank Bijleveld, who had earlier said the Russian military intelligence agency, the GRU, was the body allegedly carrying out the attempts to infiltrate cybersecurity at the OPCW.
Ms Bijleveld said the attempts took place in April 2018 but were stopped by authorities. She said four Russian diplomats were immediately expelled from the Netherlands as a result of the Dutch government's suspicions.
She also said about suspicions Russia may have attempted to hack the Dutch's investigation into the MH17 plane crash: "We have been aware of the interest of Russian intelligence services in this investigation and have taken appropriate measures."
The Malaysian Airlines flight was travelling from Amsterdam to Kuala Lumpur in 2014, when it was shot down, killing 283 passengers and 15 crew members, many of them Dutch citizens.
UK Prime Minister Theresa May and Dutch Prime Minister Mark Rutte said in a joint statement: "This attempt, to access the secure systems of an international organisation working to rid the world of chemical weapons, demonstrates again the GRU's disregard for the global values and rules that keep us all safe".
This is not the first accusation lobbed against Moscow which has resulted in an expulsion of diplomats and public condemnation.
Ms May had earlier in the year sent scores of Russian diplomats home and called the Kremlin's alleged involvement in the Salisbury attack, which also resulted in the death of UK citizen Dawn Sturgess months later, and called it a "brazen" violation of international law.
The US joined suit, sending home Russian diplomats earlier this year after a round of new sanctions was imposed for Moscow's involvement in the hacking of the 2016 US election.
Russia has been at odds with the OPCW and its members since its ruling it could determine guilt in the 4 March Salisbury attack during which former Russian spy Sergei Skirpal and his daughter Yulia were poisoned in the UK town with a military-grade nerve agent called novichok.
At the time, Moscow’s Industry Minister Georgy Kalamonov, compared the group to “the Titanic, which got a hole and began to sink” following the vote and questioned how long it would last after its expansion of powers.
The US defence secretary also said Moscow's continued deployment of nuclear-capable missiles is "untenable" and will require one of several options of response from the US and other countries.
“Russia must return to compliance with the [1987 Intermediate-range Nuclear Forces Treaty] or the US will need to match its capabilities to protect US and NATO interests," Mr Mattis said while in Brussels.
The US Ambassador to Nato, Kay Bailey Hutchinson, had earlier said if Moscow continues to be in non-compliance of the treaty then the US would need to look to ways to "take out" the weapons system.
Ms Hutchinson said the US does not itself want to violate the INF treaty, but indicated Russia could somehow compel it to do so.
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