The company's Forefront Endpoint Protection has also failed the test.
Microsoft's Security Essentials anti-virus software has failed to earn a certified status in the second round of testing by AV-Test, an independent IT security institute in Germany.
Apart from Security Essentials, Microsoft's Forefront Endpoint Protection has also failed the test.
Microsoft said: "We conduct a rigorous review of the results whenever test results warrant it. We take the protection of our customers very seriously, and the investments we make to do these reviews is an example of that commitment."
"Our review showed that 0.0033 percent of our Microsoft Security Essentials and Microsoft Forefront Endpoint Protection customers were impacted by malware samples not detected during the test.
"In addition, 94 percent of the malware samples not detected during the test didn't impact our customers."
In October last year, Microsoft failed to earn a certified status for its Security Essentials versions 4.0 and 4.1 from AV-Test.
AV-Test conducts tests every two months and the institute's test from November and December 2012 were conducted for 25 consumer antivirus security programmes.
Apart from Microsoft's products, PC Tools Internet Security 2012, AhnLab Internet Security 8.0 also failed the tests.