: Ideal dosing for the preservative-free (PF) tafluprost/timolol fixed combination (TTFC) remains to be elucidated. : This study was a prospective, observer-masked, placebo-controlled, crossover, comparison in 42 consecutive open-angle glaucoma patients whose intraocular pressure (IOP) was insufficiently controlled with preserved latanoprost monotherapy (mean 24-h IOP >20 mmHg). Patients were randomized to either morning (08:00) or evening (20:00) PF TTFC for 3 months and then crossed over. After each treatment period, patients underwent habitual 24-h IOP monitoring with Goldmann tonometry in the sitting position (at 10:00, 14:00, 18:00, and 22:00) and Perkins tonometry in the supine position (at 02:00 and 06:00). : Mean 24-h IOP on latanoprost was 22.2±3.9 mmHg. Both PF TTFC dosing regimens obtained greater reduction in mean 24-h, daytime, nighttime, and peak 24-h IOP ( < 0.001). Evening dosing provided tighter 24-h IOP fluctuation versus latanoprost ( < 0.001). Evening dosing was superior to morning dosing at four time points ( < 0.01), for the mean daytime IOP ( < 0.001) and mean 24-h IOP fluctuation ( < 0.001). Hyperemia was more common with preserved latanoprost (21.4 vs. 7.1%;  = 0.031). Patients ( = 19; 45%) preferred evening dosing. : PF TTFC provided greater 24-h IOP control and less hyperemia compared with preserved latanoprost. Evening administration of this novel medication offered superior 24-h efficacy. : Clinicaltrials.gov (NCT03612817).