An isolated bacterium, identified as Bacillus thuringiensis ITRI-G1, was able to produce active extracellular substances (denoted as AES-Bt) that exhibited efficient cell disruptive ability for a lipid-rich microalga Chlorella vulgaris CNW-11. Pretreatment of the microalga with AES-Bt for 24 h led to an increase in the extracted oil content from 34.2% to 44.3% (dry weight basis), representing an enhanced oil extraction efficiency (ηoil) of 29.3%. The ηoil value remained within the range of 26.3–33.2% when increasing the algae biomass loading from 5.0 g/l to 40.0 g/l (or decreasing AES-to-algae ratio from 200 to 25 ml/g), while the maximum ηoil value was obtained when an AES-to-algae ratio of 50 ml/g was used. Finally, reuse of AES-Bt agent was also examined. The results show that the enhanced lipid extraction efficiency slightly decreased as the number of recycling times increased. However, after being recycled and reused for four times, the AES-Bt agents could still effectively disrupt the cell wall, leading to around 92% of the original oil extraction efficiency that was obtained when the AES-Bt agents were first used. This study demonstrates that biological cell-disruptive pretreatment using the AES-Bt agents could be an effective and environmentally friendly means of increasing oil extraction efficiency with microalgae.