Nevada Athletic Commission executive Keith Kize confirmed Overeem tested unacceptably high for testosterone.
The Dutch kickboxing champion needs to win a licence from the commission if he is to face Dos Santos in the main event of UFC 146 as scheduled on May 26.
The 31-year-old, who was born in London, can request a second urine test be taken which, if returned negative, will boost his chances of gaining a licence.
But should the initial test's findings be confirmed, a new licence is unlikely to be granted and his fight with Dos Santos will not go ahead.
Overeem has previously denied allegations of steroid use and was forced to apply for a licence last December before meeting Brock Lesnar in the main event of UFC 141 following his failure to give a mandatory random sample in the build-up to the fight.
That licence was granted with a number of conditions attached to it, including one which required him to pass two random drug tests requested by the commission within six months of the Lesnar fight.