The Bulgarian Government lodged a formal objection with the European Commission, which is seeking the views of members states.
The move means the EC's reflection period for the policy will be extended by three months to late December and could delay Scottish Government plans to introduce minimum pricing next year.
The EC may ask for a more detailed justification of the policy following the objection.
A Scottish Government spokesman said the move was not unexpected and insisted ministers were confident they could demonstrate the need for minimum pricing.
However, Bulgaria's objection, based on European competition laws, was welcomed by the Wine and Spirits Trade Association (WSTA), a drinks industry body campaigning against the plans.
Its chief executive Miles Beale said: "We are not surprised that concerns about the legality of minimum unit pricing have been formally notified by the Bulgarian Government to the European Commission.
"The extended timetable for consideration of the proposals provides an opportunity for other countries to register their opposition and for the European Commission to seek an explanation from the UK Government about the legality of the Scottish Government's proposals."
The planned 50p per unit minimum price will make it illegal to sell a bottle of normal-strength wine for less than £4.69, potentially harming exporters who produce cheap alcohol.
The Scottish Government formally notified the EC of its plans after MSPs passed legislation in May. Bulgaria is the first country to formally object.
Europe-wide trade organisations representing wine and spirits producers have already registered protests.
Scottish Conservative MEP Struan Stevenson said a legal challenge to the policy was now inevitable. He said: "One of the key values of the European Union is the single market on trade and freedom of movement for people and goods.
"When a part of that member state like Scotland creates an invisible barrier for that, it results in questions which could very well end up in court.
"What this tells us is that the involvement of the European Court of Justice is inevitable. It is quite a matter for concern that the Scottish Government were desperate to plough ahead and not consider the unintended consequences of this."
Bulgarian wines are among the most affordable on supermarket and off-licence shelves.
Tesco Online was yesterday advertising a "smooth and juicy" Reka Valley Bulgarian Merlot for £19.86 per case, equivalent to £3.31 per bottle, well below the proposed minimum price.
A Scottish Government spokesman said: "Under European law, we need to show minimum pricing of alcohol is justified on the basis of public health and social grounds. We are confident it is justified in Scotland and that we will be able to demonstrate this.
"We believe minimum unit pricing is the most effective pricing measure because it targets a reduction in consumption of alcohol which is cheap relative to its strength.
"Independent modelling demonstrates the significant reduction in harm, including 60 fewer deaths and 1600 fewer hospital admissions in the first year alone, growing to a total of 300 fewer deaths annually after 10 years.
"We consider our preferred minimum unit price of 50p is justified on the basis of public health and social grounds and is proportionate.
"This development is not unexpected and within the usual procedures for notification under the Technical Standards Directive."
The Government was unclear on the possible impact of the objection on their timescale for bringing in minimum pricing.
A spokeswoman said: "We have only ever said it would be introduced in April at the earliest. It's still April at the earliest that we are looking at."
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