Tomas Kalas' first half strike ensured the Super Eagles head into the World Cup with no victory in their last four games.
Nigeria started in a similar shape to the second half against England at Wembley with John Ogu starting as the left-sided centre-back and Wilfred Ndidi playing alongside John Mikel Obi and Alex Iwobi in midfield. Gernot Rohr, seeking to further test the viability of the 3-5-2 rolled out the formation again with Victor Moses playing off Odion Ighalo in attack.
Brighton and Hove Albion defender, Leon Balogun looked in fine fettele when he embarked on a run into the attack from deep in the right centre-back position. With Shehu Abdullahi repri on his outside the
The Czechs were the better side in the opening stages as they drew a rasping save from Super Eagles goalkeeper, Francis Uzoho in the 15th minute.
Karel Jarolim's men cranked up the pressure, winning successive corners that threatened the disorganized Super Eagles backline.
The Super Eagles eventually broke; Kalas shot past Uzoho to put Czech ahead in the 25th minute from a set-piece.
After poor and nervy defending of set-pieces, the Super Eagles couldn't properly clear their lines for the Czech's fifth corner kick, leaving Chelsea player, Tomas Kalas to fire home from inside the box after Uzoho saved Tomas Soucek's header.
Rohr's men couldn't muster a response to Kalas' goal with Czech going close to getting a second. AS Roma forward, Patrick Schick dribbled his way past William Troost-Ekong into the Nigerian area but couldn't hit the target after he was through on goal.
Ighalo, who had been on the periphery of the game, almost scored an equalizer but goalkeeper Tomas Vaclik got a strong hand on the shot to turn it for a corner kick.
In search of a goal, Wilfred Ndidi's unleashed a powerful long range strike after a smart build up but Vaclik produced an equally brilliant stop to keep his team ahead.
The Super Eagles midfield looked more comfortable with anchorman Wilfred Ndidi back in the side but the Super Eagles created little chances in an unimpressive first half showing.
After half-time, the Super Eagles were more composed, patiently building possession with Iwobi, Mikel and Ndidi combining in good spells of possession football.
Nigeria was more assertive and looked to dominate the ball but the team lacked penetration to break down the sturdy Czech defence.
The game was paused briefly in the 60th minute due to thunderstorms at the Rudolf Tonn Stadium, Austria.
After resumption of play, Victor Moses missed a golden chance to put the Super Eagles level but couldn't shuffle his legs properly with the ball stuck between his legs when he got the ball in front of substitute goalkeeper Tomas Koubek.
Tyronne Ebuehi, who replaced Abdullahi at half-time, called for a penalty when he felt he was brought down in the box but the referee called for a free-kick outside the box where the foul action started.
At the other end, Uzoho saved pulled off a save of Jakub Jankto's shot in the 75th minute and two minutes later he was pulling off a stunning save with his feet from Michael Krmencik's header.
Rohr brought on gangling striker Simy Nwankwo for Ighalo but he Crotone forward couldn't make a telling impact on the game. Ahmed Musa and Kelechi Iheancho also came on in the late stages of the game but couldn't score an equalizer for the Super Eagles.
The Super Eagles are grouped alongside Argentina, Croatia and Iceland in the World Cup in Russia. The team plays its opening game against Croatia on June 16.