The defeat saw the Premier Soccer League (PSL) giants slip down to second place on the standings, while the Blood and Gold climbed to the top of the table.
Esperance were expected to come up with an offensive game plan after adopting a defensive approach during their 0-0 draw with Pirates at the Orlando Stadium over a week ago.
Attacking trio of Youcef Belaili, Taha Yassine Khenissi and Anice Badri kept their places in the starting line-up as Esperance looked to secure their second win of the group.
The Buccaneers were keen to take advantage of the fact that the game was played behind closed doors as Esperance were serving their two-match crowd ban.
Happy Jele, who missed the goalless draw against the Tunisian giants, made his return to the Bucs starting line-up and the defender was eager to lead his troops to victory in Tunis.
However, the hosts dominated the match from the start and Pirates shot-stopper Wayne Sandilands was the busier of the two goalkeepers in the opening stages of the encounter.
The 35-year-old shot-stopper, who was part of the Mamelodi Sundowns team that won the 2016 Champions League, was beaten in the 16th minute following a well-taken free-kick.
Last season's Champions League golden boot winner Badri hit the back of the net from Belaili's free-kick to make it 1-0 to Esperance as Pirates conceded their first goal in the group stages.
The visitors were struggling to create goalscoring chances for their strikers as the Blood and Gold defence which was led by experienced defender Chamseddine Dhaouadi looked solid and impenetratable.
Esperance were deservedly leading 1-0 at the interval with Pirates having failed to test Rami Jridi after the first 45 minutes.
The defending champions continued to dominate after the restart and Sandilands came to Bucs' rescue three times, denying Mohamed Yacoubi and Khenissi before the hour-mark.
Milutin 'Micho' Sredojevic reacted by introducing Zambian striker Justin Shonga, who is Pirates' top goalscorer in this season's Champions League with four goals.
The Buccaneers came into this clash having scored the majority of their goals (25.9%) between the 75th minute and 90th minute in their Caf Champions League games.
However, the 1995 champions were limited to long-range shots and hardly tested Jridi.
As they pushed for the equalizing goal, Pirates defence were badly exposed and Esperance took advantage of that.
Second half substitute Haythem Jouini punished Pirates as he scored in the 89th minute from close range to seal Esperance's 2-0 victory on the day.