Changes in the microbial populations in the conjunctival sacs of animals have traditionally been evaluated using conventional microbiology techniques. The goal of this study was to examine the suitability of a methodology which may reveal a previously unknown microbiome inhabiting feline conjunctival membranes. In the present study, we determined the microbial diversity in feline conjunctivas based on 16S rRNA gene sequence analysis. Five taxa not described earlier in veterinary ophthalmology (i.e., Staphylococcus caprae, Staphylococcus succinus, Propionibacterium acnes, Psychrobacter faecalis, and Bacillus subtilis) were identified in feline conjunctivas with a high similarity (99-100%). The study demonstrates that the feline conjunctival sacs are inhabited by much more rich and diverse microbial communities than previously thought using culture-based methods. From the clinical perspective, this could suggest that other laboratory procedures (e.g., extended incubation time in the case of Actinobacteria, formerly order Actinomycetales) or a new tool like culture-independent approaches (next-generation DNA sequencing) should be taken into account.