The nuclear plant in Temelin, which went into service in October 2000, has had numerous technical problems forcing emergency shutdowns. The anti-nuclear demonstrators said they are concerned about possible radioactive pollution.
Temelin is located about 60 kilometers from the border with Austria, which has been a non-nuclear country since 1978.
The anti-nuclear protest was organized at the Wullowitz-Dolni Dvoriste border crossing by several Austrian environmentalist groups including Anti-Atom, Stop Temelin and Atomstopp, which claim the Austrian government is not doing enough.
"The Austrian government is not exercising sufficient pressure to get a resolution to concerns about security and is losing the chance to get vast improvements at Temelin," the groups said in a joint communique.
The Czech Temelin plant, which was developed under the ex-Soviet regime and includes Russian reactors, was entirely reviewed after the fall of communism, by the US nuclear power plant builder Westinghouse, among others.