As face mask wear can result in the redirection of nasal and oral exhalation toward the ocular region, this study investigated the impact of face mask wear on the conjunctiva, eyelid margin, and contact lens (CL) surface microbiome. In this prospective, cross-over study, experienced CL wearers (N=20) were randomized to wear a face mask for 6 hr/day (minimum) for a week or no mask for a week. The conjunctiva, eyelid, and CLs were then sampled. After a 1-week washout period, participants were crossed over into the alternate treatment for 1 week and sampling was repeated. Sampling was bilateral and randomly assigned to be processed for culturing or 16S ribosomal(r) RNA gene sequencing. Culturing showed no effect of mask wear on the average number of bacterial colonies isolated on the conjunctiva, eyelid, or CL, but there was increased isolation of Staphylococcus capitis on CL samples with mask wear (P=0.040). Culture-independent sequencing found differences in the taxonomic complexity and bacterial composition between the three sites (P<0.001), but there was no effect of bacterial diversity within and between sites. Mask wear did not impact dry eye or CL discomfort, but increased ocular surface staining was reported (P=0.035). Mask wear did not substantially alter the microbiome of the conjunctiva, eyelid margin, or CL surfaces in uncompromised healthy eyes.