Purpose: Dry eye disease (DED) is a multifactorial ocular surface disorder characterized by tear film instability and chronic inflammation. Natural compounds with anti-inflammatory activity have therefore attracted increasing interest. Chelidonium majus (C. majus) latex contains bioactive alkaloids with proven anti-inflammatory properties; however, its potential role in DED has not yet been investigated. Methods: Forty-six Wistar rats were randomized into six groups: Control, DED, Loteprednol etabonate (LE), and three C. majus latex dilutions (1/1, 1/10, 1/100). DED was induced by twice-daily instillation of 0.2% benzalkonium chloride (BAC) for 14 days. Following induction, LE or C. majus latex was administered according to group allocation; controls received vehicle. Tear volume (phenol red thread test), fluorescein corneal staining score (CSS), and Cochet–Bonnet esthesiometry were measured on days 0, 14, 21, and 28. On day 29, corneal tissues were analyzed by ELISA for TNF-α, IL-1β, IFN-γ, NF-κB, MMP-2, MMP-9, TIMP-2, AQP1, and AQP5, and underwent histopathological evaluation (H&E). Latex composition was characterized using LC-QTOF-MS. Statistical analysis included ANOVA with post hoc Tukey testing; p < 0.05 was considered significant. Results: At week 2, the DED groups demonstrated reduced tear volume (2.13 ± 0.83 mm) and increased corneal staining scores (CSS) (4.63 ± 1.06). By week 4, tear secretion improved in the LE (4.38 ± 0.52 mm) and in the C. majus 1/1 (4.56 ± 0.56 mm), 1/10 (4.88 ± 0.52 mm), and 1/100 (4.56 ± 0.50 mm) groups, approaching values observed in the control group. CSS decreased markedly in the C. majus 1/10 (0.38 ± 0.52) and LE (1.13 ± 0.83) groups, whereas the DED group remained elevated (2.38 ± 0.52). Corneal sensitivity improved in the LE and C. majus 1/1 (3.44 ± 0.42 mm), 1/10 (3.75 ± 0.27 mm), and 1/100 (3.50 ± 0.46 mm) groups. Inflammatory cytokines and MMP-2/9 levels decreased significantly across treatment groups, with the most pronounced reductions observed in the C. majus 1/10 group. Histopathological analysis revealed better preservation of stromal architecture and reduced inflammatory infiltration in treated groups compared with the DED group. Conclusions: Chelidonium majus latex demonstrated significant anti-inflammatory and tissue-protective effects in this experimental model, supporting further investigation as a potential adjunctive approach in inflammatory DED.