The influence of several nonionic surfactants (Tween-20, Tween-40, Tween-60, Span-20, Span-60, or Span-80) and anionic surfactants (sodium lauryl sulfate, sodium stearoyl lactylate, and sodium stearyl fumarate) showed drastic differences in the rank order of lipase activity/lipid bioaccessibility. The biophysical composition of the oil and water interface has a clear impact on the bioaccessibility of fatty acids (FA) by altering the interactions of lipase at the oil-water interface. It was found that the bioaccessibility was positively correlated with the hydrophilic/lipophilic balance (HLB) of the surfactant and inversely correlated to the surfactant aliphatic chain length. Furthermore, the induction time in the jejunum increased as the HLB value increased and decreased with increasing aliphatic chain length. The rate of lipolysis slowed in the jejunum with increasing HLB and with increasing aliphatic chain length.