During the last few decades native European hares (Lepus europaeus) have declined in Central and Northern Italy. Despite this trend having multiple causes, it was hypothesized that invasive Eastern cottontails (Sylvilagus floridanus) contributed to the decline through apparent competition and disease transmission. In this research we explored whether cottontails may act as carriers of EBHSV (European Brown Hare Syndrome Virus) and RHDV (Rabbit Haemorrhagic Disease Virus), the viral agents of two major diseases affecting lagomorphs in Europe. We took biological samples from 267 cottontails that were shot between March and August 2015 in Tuscany, performing specific antigenic and serological ELISA tests for both viruses as well as molecular investigation for lagoviruses. Virologic tests were all negative and serological titers were below the threshold that could indicate the active circulation of either of the two pathogenic viruses. Our findings suggest that cottontails were not playing an active role as carriers or reservoirs for both known virulent lagoviruses and were also not infected with non-pathogenic lagoviruses — at least at that time in the study area