The Italian Foreign Ministry summoned France's ambassador on Saturday, after uniformed French customs officers crossed the border in the Alps to confront a man suspected of carrying drugs.
France insisted that the presence of its personnel at a railway station just inside Italy was acceptable.
However French ambassador Christian Masset was summoned over what Rome termed "a serious act considered outside the scope of cooperation between states sharing a border."
The foreign ministry added that the EU neighbours would address the issue further at a meeting in the northern city of Turin on April 16.
The Rainbow4Africa NGO, which has for months occupied part of the train station in the alpine village of Bardonecchia to aid migrants headed for France, filed a complaint after the French officials turned up there late Friday and told a Nigerian man to provide a urine sample.
Italian officials reacted with outrage, prompting an official explanatory statement from the French embassy.
Gerald Darmanin, the French minister charged with overseeing customs, signed the statement which explained how French officials came to be present when the Paris-bound train arrived from Milan.
"These uniformed agents identified as French customs officials suspected one passenger, a Nigerian national resident in Italy, of having drugs on his person.
"In line with article 60 bis of the customs code the agents asked the person if he would consent to providing a urine sample to detect drugs which he agreed to do in writing," the statement read.
"In order to carry out the test in conditions respecting the person, the agents waited until the train arrived to use the facility at Bardonecchia station which was placed at the disposal of French customs," the statement went on, saying the procedure respected current guidelines laid down in 1990.
The French officials duly carried out the test which came back negative.
But the Italian foreign ministry said France had failed to keep them fully abreast of developments, leaving Rome to slam the incident as "unacceptable."
Matteo Salvini, the head of Italy's far-right League, commented that Rome, "instead of expelling Russian diplomats should remove French diplomats," juxtaposing the incident with the furore over the recent poisoning of a former Russian spy in Britain, widely blamed on Moscow.