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Despite the high burden of vector-borne disease in (sub)tropical areas, few information are available regarding the diversity of tick and tick-borne pathogens circulating in the Caribbean. Management and control of vector-borne disease require actual epidemiological data to better assess and anticipate the risk of (re)emergence of tick-borne diseases in the region. To simplify and reduce the costs of such large-scale surveys, we implemented a high-throughput microfluidic real-time PCR system suitable for the screening of the main bacterial and parasitic genera involved in tick-borne disease and potentially circulating in the area. We used the new screening tool to perform an exploratory epidemiological study on 132 adult specimens of <i>Amblyomma</i><i> </i><i>variegatum</i> and 446 of <i>Rhipicephalus </i><i>microplus</i> collected in Guadeloupe and Martinique. Not only the system was able to detect the main pathogens of the area&#8212;<i>Ehrlichia</i><i> </i><i>ruminantium</i>, <i>Rickettsia </i><i>africae</i>, <i>Anaplasma</i><i> </i><i>marginale</i>, <i>Babesia </i><i>bigemina</i> and <i>Babesia </i><i>bovis</i>&#8212;but the system also provided evidence of unsuspected microorganisms in Caribbean ticks, belonging to the <i>Anaplasma</i>, <i>Ehrlichia</i>, <i>Borrelia</i> and <i>Leishmania</i> genera. Our study demonstrated how high-throughput microfluidic real-time PCR technology can assist large-scale epidemiological studies, providing a rapid overview of tick-borne pathogen and microorganism diversity, and opening up new research perspectives for the epidemiology of tick-borne pathogens.