N-trimethyl chitosan (TMC) polymers differing in quaternization degree (QD) and molecular weight (MW) were prepared from two chitosans 90% deacetylated, one of higher MW (1460 kDa) (TMCH), the other of lower MW (580 kDa) (TMCL), by one (TMCH1, QD = 4%; TMCL1, QD = 3%), two (TMCH2, QD = 35%; TMCL2, QD = 46%), or three (TMCH3, QD = 90%; TMCL3, QD = 78%) reductive methylation steps. The derivatives were tested and compared for their ability to enhance the permeability of ofloxacin across rabbit corneal epithelium, reconstituted in vitro. TMC polymers of intermediate QD (TMCH2 and TMCL2), at the concentration of 0.001% w/v, produced significant permeability enhancements, independent of polymer MW. The enhancing effect did not increase when QD was increased (TMCH3 and TMCL3), while it was not significant with low QD values (TMCH1 and TMCL1). Such an effect was specific of chitosan derivatives, because fully quaternized DEAE-dextran (MeDD) was ineffective. The transcorneal permeability-enhancing property of TMCH2 and TMCL2, and the inefficacy of MeDD were confirmed by in vivo tests on rabbit eyes. However, unlike the in vitro experiments, the in vivo ones showed a stronger effect of the TMC having higher MW. TMCH2 produced antibiotic levels in the aqueous humor higher than the MIC(90%) for the more resistant ocular pathogens. These results point to this derivative as a potential ofloxacin absorption enhancer for the topical treatment of endophthalmitis.