The purpose of this study was to evaluate the impact of free fatty acids (FFA), namely oleic (OA) and linoleic (LA) ones, on meibomian lipid films (MLF) using a Langmuir trough (LT) and a Brewster angle microscope (BAM). Human meibum was collected from healthy volunteers. A Tris-buffered saline (TBS, pH 7.4) was used as the control aqueous subphase for LT experiments. Then, varying amounts of OA and LA were dissolved in TBS to make FFA-containing subphases. Predetermined amounts of meibum were loaded onto the surface of the (TBS/±FFA) subphases to form MLF. Then, surface pressure-area (π/A) isotherms of MLF were recorded. Standard rheological parameters such as rigidity, elasticity, and hysteresis, were computed. In a separate experiment, OA and LA were pre-mixed with meibum at different weight ratios prior their spreading onto the control TBS subphase, and the (π/A) isotherms of the resulting mixed films of meibum and FFA were studied and analyzed in the same fashion as described above. When studied at the normal corneal temperature of 34 °C with the (TBS/-FFA) subphase, meibum formed stable films. When (TBS/+FFA) subphase was used, both FFA quickly disrupted the MLF, acting in a similar fashion. BAM revealed that the most dramatic changes in the structure of MLF occurred in the range of OA concentrations between 5 and 15 μM. However, this effect was apparent even with 2.5 μM OA. When OA was pre-mixed with meibum, but was absent from the subphase, it caused gradual concentration-dependent changes in the (π/A) isotherms, but the MLF did not disappear from the surface. Thus, tested FFA showed a remarkable ability to disrupt, and/or prevent the formation of, human MLF, which could contribute to the onset of those forms of dry eye disease that are associated with enhanced activity of lipolytic enzymes, such as chronic blepharitis.