Microalgal phospholipid bilayer contributes to the molar excesses of methanol and high acid concentration required in reactive extraction to achieve high fatty acid methyl ester (FAME) yield. This study reports an investigation into the effects of pre-soaking Nannochloropsis occulata in methanol at 600:1 and 1000:1 methanol to oil molar ratios prior to acid-catalyzed in situ transesterification at 8.5:1 and 15:1 H₂SO₄ to oil molar ratios on the FAME yield. The results showed that the pre-soaked Nannochloropsis occulata produced a higher FAME yield at the two tested methanol to oil molar ratios and acid concentrations than the un-soaked, resulting in a reduction in methanol volume and acid concentration. A maximum FAME yield of 98.4% ± 1.3% was obtained for the pre-soaked Nannochloropsis occulata at 1000:1 methanol to oil molar ratio and 15:1 H₂SO₄ to oil molar ratio. Both the phosphorus mass balance and conversion of the isolated phospholipids into FAME revealed that pre-soaking solubilizes the phospholipid bilayer to some degree, and contributes to an increased FAME yield.