The study investigated biochemical changes in meibomian gland (meibum) secretions in patients with obstructive meibomian gland dysfunction (MGD) using Raman spectroscopy. The analysis showed that the meibum of MGD patients contains less unsaturated lipids compared to healthy subjects, which was expressed by the lower intensity ratios of I1659/I1438 and I3013/I2860 bands. Ratios reflecting the balance of lipids and proteins (I1659/I1161 and I3013/I1161) were, in contrast, higher in MGD, indicating a relative decrease in proteins in the meibum. Changes in the ratio of symmetric to asymmetric lipids (I2888/I2860) were also significant, although with a small absolute effect. Raman mapping revealed a heterogeneous spatial distribution of lipids and proteins in the meibum of MGD patients. The results suggest a disruption of the lipid composition and microstructural organization of the meibum in MGD. The study confirms that Raman spectroscopy is a useful tool for meibum analysis and detection of pathological changes in meibomian gland dysfunction.