The missing men all worked for Mighty Current, known for books critical of Beijing which closely monitors and controls dissenting voices.
"The continuing lack of information about the well-being and whereabouts of five Hong Kong residents... Associated with the publishing house Mighty Current is extremely worrying," the EU's external affairs arm said in a statement.
It noted that two of the missing were EU citizens, naming Gui Minhai as a Swedish national who went missing in Thailand in October, and Lee Po as a British national.
"This would be inconsistent with the 'one country two systems' principle," it said, referring to the accord Britain agreed with Beijing before Hong Kong returned to Chinese rule in 1997 to ensure the territory enjoyed greater freedoms than the mainland for 50 years.
Also Read
"The relevant authorities in Thailand, China and Hong Kong should investigate and clarify the circumstances of the disappearances in conformity with the rule of law," the statement added.
In 2014, tens of thousands of protestors brought parts of Hong Kong to a standstill for more than two months after Beijing imposed restrictions on candidates for the city's next leader.