Purpose. To investigate the association between precorneal tear film (PCTF)– and meibum-derived (O-Acyl)-omega-hydroxy fatty acids (OAHFAs) and PCTF thinning in meibomian gland health and dysfunction. Methods. Of 195 eligible subjects (18–84 years, 62.6% female), 178 and 170 subjects provided both PCTF optical coherence tomography (OCT) imaging and mass spectrometry data for tears (n = 178) and meibum (n = 170). The PCTF thinning rate was measured in the right eye using an ultra-high-resolution, custom-built OCT. Tear and meibum samples from the right eye were infused into the SCIEX 5600 TripleTOF mass spectrometer in the negative ion mode. Intensities (m/z) of preidentified OAHFAs were measured with Analyst 1.7TF and LipidView 1.3 (SCIEX). Principal component (PC) analyses and Spearman’s correlations (ρ) were performed to evaluate the association between OAHFAs and PCTF thinning rates. Results. In meibum and tear samples, 76 and 78 unique OAHFAs were detected, respectively. The first PC scores of the meibum-derived OAHFAs had statistically significant correlations with PCTF thinning rates (ρ = 0.18, P = 0.016). Among 10 OAHFAs with the highest first PC loadings, six OAHFAs had negative correlations with PCTF thinning rate (18:2/16:2, ρ = −0.19, P = 0.01; 18:2/30:1, ρ = −0.21, P = 0.008; 18:1/28:1, ρ = −0.22, P = 0.004; 18:1/30:1, ρ = −0.22, P = 0.005; 18:1/25:0, ρ = 0.22, P = 0 .006; and 18:1/26:1, ρ = −0.22, P = 0.006), while one OAHFA had a positive correlation with PCTF thinning rate (18:2/18:1, ρ = 0.48, P = 0.006). Tear film-derived OAHFAs had no association with the PCTF thinning rate. Conclusions. Several human meibum-derived OAHFAs showed significant associations with PCTF thinning, suggesting that these OAHFAs could be implicated in the mechanism underlying the stabilization and thinning of the PCTF. The tear-film derived OAHFAs were, however, independent of the rate of PCTF thinning.