During the brief conversation, Obama made special mention of the families of the victims who died in the attack in Madrid on March 11th 2004.
On that day, a series of bombs exploded within minutes aboard four packed commuter trains heading for Madrid's Atocha station. The coordinated attack — claimed by militants who said they had acted on Al-Qaeda's behalf over Spain's role in the US-led invasion of Iraq — killed 191 people and wounded about 2,000.
Monday's conversation also saw Rajoy and Obama speaking about the crisis in Ukraine sparked by what some have called a hostile Russian takeover of Crimea on the Black Sea.
The move has been condemned by the European Union and the US, with economic sanctions against Russia being touted.
Obama "welcomed the strong, unified stance of the US and the EU regarding Russia’s military intervention in Ukraine," according to a statement released by the US embassy in Madrid.
Both leaders "reiterated their grave concern over Russia’s clear violation of international law and reaffirmed their support for Ukraine’s sovereignty and territorial integrity," the statement continued.
Rajoy and Obama also "welcomed the restraint shown by the Ukrainian government in responding to Russian violations of Ukrainian sovereignty".
They called for direct dialogue between Ukraine and Russia and rejected a planned March 16th referendum in Crimea as "a violation of Ukraine's constitution", according to the US embassy.