The court ruled in favour of researchers who say human embryonic stem cell research involved the destruction of human embryos.
Judge Royce Lamberth said lawsuits brought against the new guidelines could now go ahead.
President Obama lifted a ban on funding for stem cell research in March.
Critics say the ban, which was kept in place by Mr Obama's predecessor, George W Bush, impeded the fight to find treatments for diseases like Alzheimer's, Parkinson's and diabetes.
The suit, which was also backed by some Christian groups, is against the National Institutes of Health (NIH). Those against the new plan to increase funding argued the NIH policy violated US law and also took funds from researchers seeking to work with adult stem cells.
"ESC (embryonic stem cell) research is clearly research in which an embryo is destroyed," Judge Lamberth said.
He added: "To conduct ESC research, ESCs must be derived from an embryo. The process of deriving ESCs from an embryo results in the destruction of the embryo. Thus ESC research necessarily depends upon the destruction of a human embryo."
But Judge Lamberth said an injunction would not "seriously harm" the embryonic studies because it did "not interfere with their [researchers'] ability to obtain private funding for their research".
The Dickey-Wicker Amendment, which Congress adds to budget legislation each year, played an important role in the case. The amendment bans the use of federal funds to destroy human embryos.
With the injunction in place, Judge Royce Lamberth is now likely to hear groups on both sides of the case argue whether the new guidelines should be permanently blocked or allowed to continue.
A US district court has issued a temporary injunction blocking plans by the Obama administration to increase funding for stem cell research.