BRUSSELS—Euroskeptic and nationalist politicians who performed well in recent European Parliament elections appear unlikely to wield significant power in the legislature after they failed to organize as a single group.

The nationalist parties of French opposition leader Marine Le Pen and Italy’s Deputy Prime Minister Matteo Salvini renewed their alliance Thursday in the European Union’s parliament, but fell short of their goal of uniting all euroskeptic factions into one powerful caucus.

Nativist, anti-immigrant and euroskeptic parties gained in elections for the European Parliament in May. Ms. Le Pen and Mr. Salvini campaigned on uniting the continent’s skeptics into a force able to thwart the EU from within.

Disagreements over Russia and personal animosities have scuttled their ambition. Since the elections, the Le Pen-Salvini alliance that already existed in the EU parliament succeeded in peeling off only a dozen lawmakers from other euroskeptic groups.

Instead of becoming the second-largest caucus in the 751-strong body, Mr. Salvini’s “Identity and Democracy” group is relegated to fifth position, behind pro-European factions that hold a two-third majority.