Purpose: This study evaluated the presence and roles of cholesteryl esters (CEs) and wax esters (WEs) from human tear film and meibum in meibomian gland dysfunction (MGD). Methods: Out of 195 enrolled subjects, 164 and 179 subjects provided tear and meibum samples, respectively. Subjects were classified into normal, asymptomatic MGD, MGD, and mixed (MGD & aqueous deficient). The precorneal tear film (PCTF) thinning rate (evaporation) was measured using optical coherence tomography. Lipids extracted from tear and meibum samples were infused into a SCIEX 5600 TripleTOF mass spectrometer. CE and WE intensities quantified with Analyst 1.7 TF and LipidView 1.3 were compared across disease groups in MetaboAnalyst 5.0 and correlated with PCTF thinning rates. Results: The numbers of unique CEs and WEs identified in the samples were 125 and 86, respectively. Unsupervised Principal Component (PC) analysis and supervised Partial Least Square Discriminant analysis exhibited little separation among groups for both CEs and WEs in tears and meibum. Spearman's correlation analyses showed no association between either the first or second PC scores with PCTF thinning rates. Conclusion: The abundances of human PCTF and meibum-derived CEs and WEs were independent of MGD disease status and PCTF thinning (evaporation). CEs and WEs alterations do not contribute to alterations in tear film dynamics in MGD, such as has been demonstrated by the (O-acyl) ω-hydroxy fatty acids (OAHFAs).