Travelling supporters arrived in the Portuguese city earlier this week ahead of Gareth Southgate's side taking on Netherlands in the semi-finals of the inaugural Nations League Finals on Friday (AEST).
After an instance of anti-social behaviour outside a bar, riot police charged England fans when bottles were thrown at a Porto fan zone on Avenida dos Aliados, where Portugal fans were gathering to watch their team's 3-1 win over Switzerland.
Deputy Chief Constable Mark Roberts, football policing lead at the National Police Chiefs' Council said: "Earlier this evening Portuguese police deployed riot officers against a group of England fans in the main square area of Porto.
"It is believed the fans had been throwing bottles at Portugal fans who were watching the match in the same area.
"This is the second evening in a row where disorder has occurred in Porto. Last night there were issues outside a bar, where bottles were thrown and minor damage was caused. It is completely unacceptable."
The incidents come as a blow to the Football Association (FA), which launched a campaign video entitled 'Don't Be That Idiot' last week that sought to urge fans against being involved in scenes such as those witnessed in Porto.
FA head of security Tony Conniford expressed concern about a "stag-do culture" among an element of the travelling England support for games in more easily accessible destinations such as Portugal.
"A lot of it is alcohol-fuelled and there almost becomes an acceptance that because you are at the football, anything goes," Conniford, formerly assistant director at the UK Football Policing Unit, said when the campaign was launched.
"I find that difficult to buy into and people need to have a look at themselves and start to think, 'If my relatives, wife or children were here with me, would it be an enjoyable experience?' And the answer is 'no'."
"I don't want to stereotype people and put everybody into a box. That is very dangerous. But it would be stupid to say we had not recognised groups of young, adult males booking together are the ones we are more concerned about.
"It's that holiday, stag-do culture where young guys get together and suddenly anything goes."
Around 15,000 England fans are expected to be on the ground for the game against Netherlands at Estadio D. Afonso Henriques, which has a tournament capacity of 26,500.
The Three Lions will return to Guimaraes on Monday (AEST) to face Switzerland if they lose to Ronald Koeman's side, while victory would bring a final against Portugal in Porto later that day with up to 18,000 England supporters estimated to be in attendance by the weekend.