In other preliminary round action, New Zealand beat South Korea 81-73 and Japan came through in the final five minutes to beat Nigeria 79-73.
The U.S. women are considered heavy favourites to win their third consecutive Olympic gold.
But after watching the American men on Sunday suffer their first defeat in Olympic play since 1988, no one was taking anything for granted against a talented Czech side that placed second in the 2003 European championship.
"Any time you watch the men's team get beat, you want to make sure it doesn't also happen to you. I probably had a little more fire than I usually would because I didn't want to experience that tonight," said U.S. coach Van Chancellor.
The Czechs came out strong and added to Chancellor's worries, taking a 21-18 lead after the first quarter.
But Swin Cash sparked a U.S. comeback and the Americans pulled away with a 14-0 run in the second quarter.
Lisa Leslie and Tina Thompson dominated for the U.S., who scored a total of 46 points under the basket.
"We started off a little slow, and the Czech team was really strong offensively," Thompson said.
"Our inside game has been very strong, Lisa has created quite a presence," she added.
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