The Mesenchymal Stromal Cells from umbilical cord Wharton's jelly (WJSCs) are a source of cells with high potentiality for the treatment of human immunological disorders. Footprints of the oncogenic viruses Simian Virus 40 (SV40) and JC Virus (JCPyV) have been recently detected in human WJSCs specimens. The aim of this study is to evaluate if WJSCs can be efficiently infected by these Polyomaviruses and if they can potentially exert tumoral activity. Cell culture experiments indicated that WJSCs could sustain both SV40 and JCPyV infections. A transient and lytic replication was observed for JCPyV, while SV40 persistently infected WJSCs over a long period of time, releasing a viral progeny at low titer without evident cytopathic effect (CPE). Considering the association between SV40 and human tumors and the reported ability of the oncogenic viruses to drive the host innate immune response to cell transformation, the expression profile of a large panel of immune mediators was evaluated in supernatants by the Bioplex platform. RANTES, IL‐3, MIG, and IL‐12p40, involved in chronic inflammation, cells differentiation, and transformation, were constantly measured at high concentration comparing to control. These findings represent a new aspect of SV40 biological activity in the humans, highlighting its interaction with specific host cellular pathways. In view of these results, it seems to be increasingly urgent to consider Polyomaviruses in the management of WJSCs for their safely use as promising therapeutic source. J. Cell. Physiol. 9999: 1–7, 2016. © 2016 Wiley Periodicals, Inc. This study aims to evaluate if WJSCs can be efficiently infected by the Polyomaviruses SV40 and JCPyV and if they can exert their potential tumoral activity. Results reported a transient and lytic replication for JCPyV, while SV40 persistently infected WJSCs over a long period of time, releasing viral progeny. Specific factors, including RANTES, IL‐3, MIG, and IL‐12p40, involved in chronic inflammation, cells differentiation, and transformation, were constantly produced at high concentration. These findings represent a new aspect of SV40 biological activity in human host. In view of these results, it seems to be increasingly urgent to consider Polyomaviruses in the management of WJSCs for their safely use as promising therapeutic source.