"Vertically integrated network operators should not discriminate against competitors in favour of their own downstream business," Kroes told telco lobbyists at a conference in Brussels on Tuesday (30 November).
"We will crack down if some choose to mess up the playing field for others," she warned.
The commissioner has been trying to create a level playing field in the market by allowing smaller network providers to access the 'local loop' – the last stretch of wire between telephone exchanges and people's homes that are generally owned by incumbent firms.
"We are kidding ourselves if we think that non-discrimination remedies have fully eradicated discriminatory behaviour of dominant operators," Kroes, who is a former competition commissioner, continued.
"I urge national regulators not to hesitate to take action against any company found doing this," she added.
The warning comes just one month after Telecom Italia revised its wholesale access prices from 24% to 9.4% after it received a letter from Kroes on 21 October.
Dominant firms have been reluctant to lower their prices, arguing that charging infrastructure to smaller firms is important to spur investments in next generation high-speed fibre networks.
"If the charges for copper are too low, we harm investment in fibre," an industry source explained.
ETNO, an association representing incumbent telecoms operators, said the commissioner had recognised the importance of attracting investment in fibre.
"As highlighted by Vice-President Kroes today, there is a risk that artificially reduced wholesale charges for copper networks will erode the price level on broadband markets, making fibre products less attractive to invest in," the group said in a statement.
In September, the commissioner unveiled plans to regulate access to high-speed networks, the EU executive's so-called Next Generation Networks (NGN) recommendation.
As its name indicates, the recommendation is not legally-binding. Member states are invited to follow the Commission's advice, but there is little chance of forcing a recalcitrant national regulator to implement it.