Alfonzo also tested positive in 2008 for performance-enhancing drugs.
The catcher said he would appeal the suspension through the Major League Baseball Players Association.
"I am surprised by this positive test," he said. "I learned my lesson in 2008 and have not taken any prohibited substances since then. With the union's help, I intend to fight this suspension and look forward to appearing before the arbitrator in the near future."
The union said it would "do its best to preserve and protect the rights granted to Eliezer under the terms of the Joint Drug Agreement."
Alfonzo, batting .267 in 25 games for the Rockies, is the second MLB player ever to face a 100-game suspension. Manny Ramirez also was slapped with the penalty in April.
The former Boston Red Sox slugger, then playing for Tampa Bay, retired rather than do the time for his second steroids violation.