PCR amplification was applied to screen the presence of both Borrelia burgdorferi s.l. and Ehrlichia species in pools of field-collected Ixodes ricinus ticks. The specimens so far analysed (n = 55), grouped in 11 pools, were sampled in Feltre area (Veneto region, NE Italy). Five pools proved positive for B. valaisiana (45%) and one of them (9%) was also positive for Ehrlichia, that was further characterised as a HGE-like Ehrlichia. This is the first report of the two bacteria in the Veneto region. The pool positive for both pathogens was used to adjust a multiplex PCR assay, which allowed the detection and identification of both parasites in a single experiment. The advantages offered by this assay, when standardised, will substantially broaden the perspectives of ecological and epidemiological investigations on animal/human Lyme disease and ehrlichiosis, greatly facilitating disease surveillance and control programs.