Abstract Background Cancer is a leading cause of mortality for both humans and dogs. As spontaneous canine cancers appear to be relevant models of human cancers, developing new therapeutic approaches could benefit both species. Oncolytic virotherapy is a promising therapeutic approach in cancer treatment. TG6002 is a recombinant oncolytic vaccinia virus deleted in the thymidine kinase and ribonucleotide reductase genes and armed with the suicide gene FCU1 that encodes a protein which catalyses the conversion of the non-toxic 5-fluorocytosine into the toxic metabolite 5-fluorouracil. Previous studies have shown the ability of TG6002 to infect and replicate in canine tumor cell lines, and demonstrated its oncolytic potency in cell lines, xenograft models and canine mammary adenocarcinoma explants. Moreover, 5-fluorouracil synthesis has been confirmed in fresh canine mammary adenocarcinoma explants infected with TG6002 with 5-fluorocytosine. This study aims at assessing the safety profile and viral shedding after unique or repeated intramuscular injections of TG6002 in seven healthy Beagle dogs. Results Repeated intramuscular administrations of TG6002 at the dose of 5 × 10 7 PFU/kg resulted in no clinical or biological adverse effects. Residual TG6002 in blood, saliva, urine and feces of treated dogs was not detected by infectious titer assay nor by qPCR, ensuring the safety of the virus in the dogs and their environment. Conclusions These results establish the good tolerability of TG6002 in healthy dogs with undetectable viral shedding after multiple injections. This study supports the initiation of further studies in canine cancer patients to evaluate the oncolytic potential of TG6002 and provides critical data for clinical development of TG6002 as a human cancer therapy.