Previously, a quaternary ammonium (N +)–chitosan (Ch) conjugate (N +(60)–Ch) characterized by short pendant chains, made of 1.7 ± 0.1 adjacent diethyl-dimethylene-ammonium groups, substituted onto the primary amino group of the chitosan repeating units (degree of substitution, 59.2 ± 4.5%) was used to synthesize a multifunctional non-cytotoxic thiomer (N +(60)–Ch–SH(5)), carrying 4.5 ± 0.7% thiol-bearing 3-mercaptopropionamide besides quaternary ammonium groups. The present work was aimed at evaluating the potential of N +(60)–Ch–SH(5) and N +(60)–Ch as bioactive excipients for dexamethasone (DMS) eyedrops. The DMS permeability across excised rabbit cornea was enhanced over the control value by the thiomer and the parent polymer to about the same extent (3.8 vs. 4.1 times). The mean precorneal retention time and AUC in the aqueous of DMS instilled in rabbit eyes via eyedrops were enhanced by the thiomer (MRT = 77.96 ± 3.57 min, AUC = 33.19 ± 6.96 μg ml −1 min) more than the parent polymer (MRT = 65.74 ± 4.91 min, AUC = 21.48 ± 3.81 μg ml −1 min) over the control (MRT = 5.07 ± 0.25 min, AUC = 6.25 ± 0.65 μg ml −1 min). The quaternary ammonium ions were responsible for both permeabilization of corneal epithelium and polymer adhesion to precorneal mucus, while the thiols increased the latter. This synergistic action is the basis of the higher thiomer bioactivity in vivo. A good ocular tolerability of the chitosan derivatives resulted from in vivo experiments.