Mr Obama told the president on Monday that Russia should use its influence to get separatists in the country to stand down.
Armed pro-Russian separatists seized more buildings in eastern Ukraine earlier in the day, after the government in Kiev failed to follow through on a threatened military crackdown.
The White House said Mr Obama told Mr Putin that all irregular forces in the country need to lay down their arms and Russian troops needed to withdraw from Ukraine's border to defuse tensions, Reuters reports.
The Kremlin said the Russian leader said Russia was not interfering in Ukraine and urged Washington to use its influence to prevent bloodshed.
The White House says Mr Obama made clear that the diplomatic path was America's perferred way ahead, but expressed the view that Russia's current actions were neither consistent nor condusive to that, the BBC reports.
Russia said Mr Putin used the telephone call to blame the Ukranian government for pro-Russian protests in the south-east of the country, saying they stemmed from the interim administration's inability to take into account the interests of the Russian speaking population there.
Earlier, US officials stopped short of announcing a new set of sanctions against Russia, but said they were in consultations with European partners about the prospect.
The European Union agreed on Monday to step up sanctions against Moscow by expanding a list of people subjected to asset freezes and visa bans.
A senior administration official described the call between Mr Obama and Mr Putin as "frank and direct," a diplomatic construction that usually means tense.
The next round of US sanctions, which would be the fourth imposed since the Ukraine crisis began, is likely to target Russians close to Mr Putin as well as Russian entities, sources familiar with the discussions said on Sunday.
US State Department spokesperson Jen Psaki noted that the US was prepared to impose sanctions on individuals and entities in the financial services, energy, metals, mining, engineering and defence sectors.
The sanctions have been the most visible sign of US anger at Russia's annexation of the Crimea region in southern Ukraine in March, reflecting the deepest plunge in US-Russian relations since the Cold War. US officials declined to identify a timeline on Monday for further sanctions.
Ultimatum fails to stop attacks
Pro-Russian activists have attacked another building in eastern Ukraine, as separatists ignore a deadline to end their occupation of government buildings there.
About 100 pro-Russian separatists stormed the police headquarters in the city of Horlivka on Monday, while pro-separatist forces say they are in control of a small airfield in Slaviansk, about 150km from the Russian border.
Video footage on Ukrainian television has showed an ambulance treating people who were apparently injured during the Horlivka attack. In all, separatists have seized government buildings and security facilities in 10 cities, Reuters reports.
Meanwhile, Ukraine's acting president Oleksandr Turchynov has called for United Nations peacekeepers to be deployed in the east after his deadline for the rebels to lay down their arm passed with no sign of compliance.
Three European countries - Luxembourg, Lithuania and Sweden - say they support Ukraine using force to eject the separatists from government offices. European Union foreign ministers are discussing the crisis in Luxembourg.
The European Union has threatened Russia with more sanctions over its actions in eastern Ukraine, although some EU states say diplomacy should be given more time.
The US has also warned that it is likely to impose further sanctions on Russians close to the Kremlin if the escalation in eastern Ukraine continues.