Didier Deschamps made sweeping alterations for the Group D finale at Education City Stadium after Les Bleus became the first side to seal their place in the round of 16.
France still had the quality to win but ex-Sunderland man Khazri's moment of magic secured Tunisia a 1-0 victory - their first World Cup win against European opponents after 11 previous attempts.
Wednesday's result will live long in the memory but Mathew Leckie's strike 14 miles away proved a knockout blow for Jalel Kadri's side as Australia progressed instead.
Antoine Griezmann looked to have landed another gut punch when equalising deep in stoppage time, only for the goal to be disallowed for offside after the VAR's intervention.
Tunisia's superb fans showed their appreciation to their players at full-time in Al Rayyan, where France topped Group D despite Wednesday's shock loss.
The North African side came into this match in hope more than expectation, but just eight minutes in they began to believe.
Nader Ghandri excellently turned in a free-kick that would be ruled out for offside, yet that moment appeared to give Tunisia and their fans hope.
France had quality on the pitch but struggled to click, with Kingsley Coman missing the target at the end of one of their rare moves of fluidity.
Tunisia continued to look the bigger threat, with Anis Slimane seeing a blocked header easily saved before Steve Mandanda unconvincingly dealt with a Khazri effort.
The 31-year-old was looking most likely to open the scoring and went down in the box just minutes into the second half following a challenge by Aurelien Tchouameni.
Nothing was given as Tunisia continued to push, with Aissa Laidouni rifling over from an acute angle before getting the assist for a 58th-minute goal that was all of Khazri's making.
The skipper span as he collected the ball just under 50 yards from the French goal and drove forwards at pace.
First he brushed off the attention of Youssouf Fofana, before rounding Raphael Varane and getting away a low effort before Axel Disasi could tackle him, sending the ball rolling into the bottom corner.
It was an incredible moment and Khazri's last action of the evening as he was immediately replaced.
The volume inside the ground soon dropped as Tunisian fans heard Australia had taken the lead, then saw star Kylian Mbappe introduced from the bench.
Griezmann and Ousmane Dembele soon followed him on, with the latter seeing an attempt from distance saved by Aymen Dahmen.
Substitute Adrien Rabiot's volleyed effort went wide and Mbappe tried his luck from a tight angle, before Randal Kolo Muani saw a strike whistle narrowly wide.
Tunisia's substitutes huddled around a phone in stoppage time hoping for positive news from the Australia v Denmark match, but there was no late drama in that game.
There was in Al Rayyan, though.
Griezmann met a loose ball with a volley that deflected in cruelly as France looked to have secured a late draw.
The final whistle went, only for the VAR to advise a pitchside review and offside was given.