Background: Blepharitis is a very common disease in the ophthalmologic practice generally taking a chronic course with intermittent exacerbations. Several studies have linked the presence of Demodex folliculorum with chronic blepharitis, since the mite has the capacity to perpetuate the follicular inflammatory process. The prevalence of infection by Demodex spp. is variable depending on the population. In Paraguay, information on the frequency of the infestation in patients with chronic blepharitis is not available. Aim: To determine the frequency of Demodex spp, and the ocular microbiota in patients with chronic blepharitis attending the Department of Ophthalmology at the Teaching Hospital of the National University of Asuncion. Patients and Methods: Consecutively, 28 patients with chronic blepharitis, who agreed to participate in the study, were included. Eyes lashes from the upper and lower eyelids were extracted for immediate mite search by direct observation under a light microscope. Samples from eyelids were taken with Kimura spatula and then cultured on blood agar and in enrichment media and incubated in 5% CO2 at 35° C for 72 hours. Results: Among participants, females were more frequent (64%), the age ranged from 17 to 87 years (mean: 38.0; SD: ±13.5 years). The prevalence of Demodex sp was 54%. Bacteria were isolated 92.9% of cases, most frequently coagulase-negative staphylococci (75%). No association was found between socio-demographic or clinical characteristics and the presence of Demodex sp. Conclusion: The observed high prevalence of infestation by Demodex spp in patients with chronic blepharitis is consistent with other studies.