Spread by infected galleys coming from Kaffa (Crimea), the Black Death reached Genoa, as it now seems, in the late summer of 1347 AD. Genoa functioned as an epicentre from which the contagion was spread into the mainland through a complex system of routes, which linked Liguria to northern and central Italy. Along these routes various institutions were found, namely 'ospitali' (hospitals) and 'stationes' (stations), where traders and pilgrims stopped to rest and recuperate. In 2006 a multiple burial archaeologically dated to the second half/end of the 14th century was discovered in the cemetery pertaining to the 'ospitale' of San Nicolao (Genoa). The excavation showed that it contained the remains of four individuals: a 38-40 week pregnant woman with her fetus and two sub-adults. Stratigraphy showed that these individuals were buried simultaneously. Given that the dating of the burial fits the arrival of the Second Pandemic in Europe, it was hypothesized that they might have died during the Black Death epidemic. The identification of Yersinia pestis F1 antigen in three of four individuals corroborated this hypothesis. Here we report the first evidence of Y. pestis infection in 14th-century Liguria and discuss the possible mechanisms of plague dissemination from Genoa into the surrounding regions. In fact, the 'ospitale' of San Nicolao, located at 792 m a.s.l. into the Bracco Massif, was used as a resting place/hostel by traders and travellers (e.g. pilgrims heading for Rome). This 'ospitale' represented a key point leading into a system of pathways forming the initial part of the Vie Romee better known under the name of Via Francigena in the Italian territory and, as a consequence, was the ideal site from which plague could be disseminated.