They were heard yelling for the Act New Zealand leader to "bugger off" and that he was "not welcome here".
The protest was organised by the International Socialist Organisation and included a variety of groups who were vocal about issues including the Regulatory Standards Bill, pay parity, worker rights, Māori rights and the environment.
The protest took place outside the Distinction Dunedin Hotel where Act hosted a public meeting with Mr Seymour at 6pm.
Police warned advancing demonstrators to stay away from the front of the hotel in Liverpool St and to stick to the footpath.
Protest organiser Oscar Bartle said Mr Seymour should be ashamed of himself.
"How can you live with yourself when you’re destroying working-class livelihoods."
Mr Bartle said the crowd had gathered on short notice to oppose Mr Seymour scraping fair-pay agreements, his silence on the genocide in Palestine and his pushing of the Regulatory Standards Bill.
Mr Bartle said the Bill was nonsense.
Otago Staff and Students for Palestine, Green Party supporters and unionists were present at the protest.
Mr Seymour had previously said the principles of the Bill "focused the effect of legislation on existing interests and liberties".
"In a high-cost economy, regulation isn’t neutral — it’s a tax on growth.
"This government is committed to clearing the path of needless regulations by improving how laws are made." — additional reporting RNZ
