The Czechs have endured a stuttering qualifying campaign under Bilek, beating only Malta and Armenia as they struggled to find the net for a majority of their Group B games.
"I have not met the goal we have set for ourselves and I offer my resignation," Bilek said in a statement.
Bilek was never popular with fans due to a string of unconvincing results and conservative tactics but he did gain some success when he guided the Czechs to the Euro 2012 quarter-finals in Poland and Ukraine.
The former midfielder, who scored 11 goals in 35 internationals and played at the 1990 World Cup, took over four years ago after the side failed to qualify for the 2010 World Cup finals.
Despite some initial problems, Bilek ensured the Czechs kept up their record of qualifying for every European Championship since the Czech Republic and Slovakia split in 1993.
In Tuesday's match against Italy in Turin, it looked as if the Czechs might pull off an upset and keep their World Cup hopes alive after they took a 1-0 lead into the second half.
However, two goals within three minutes secured qualification for the hosts and left the Czechs four points adrift of Bulgaria, who occupy second place in the group.
Sitting on nine points from eight matches, the Czechs must win their last two games away to Malta and Bulgaria to have any chance of a securing a playoff spot for finishing second.
The Czech FA indicated that it would select one of Bilek's assistants to lead the side for the two remaining qualifiers before a permanent coach would be installed.