It is the first time violent assaults have been recorded against participants since 2011, organisers said.
Croatian police have detained several people over incidents during and after this weekend's Pride march in the capital of Zagreb.
The incidents included verbal and physical attacks on some participants and the burning of a rainbow-colored LGBT flag, the Zagreb police said Sunday. Police are still looking for some of the perpetrators.
Franko Dota, from the Zagreb Pride organization, told the state HRT television that the incidents were the first since 2011. Saturday's march was the 20th held in the Croatian capital.
“We expect the police to conduct a thorough investigation and treat all the incidents as hate crimes over sexual orientation, and not as violations of public orders,” he said.
The incidents involved anti-gay messages, a man blocking passage to two women, verbally assaulting them, and spitting at one of the women before fleeing, as well as physical attacks, police said.
In the worst attack late Saturday, a group approached two young men, one of whom was hit in the face, and a rainbow flag was snatched from him and burnt before a young woman hit both men in their faces.
Croatia is a predominantly conservative Catholic nation that joined the European Union in 2013.