The prevalence of obesity within the human population is escalating globally yearly. Obesity constitutes a complex ailment with diverse etiological factors. Recently, the infectious side of obesity aetiology, implicating pathogens such as human adenovirus 36 (HAdV-D36), has gained attention. Research indicates that HAdV-D36 can infect humans and various animal species, including mice, monkeys, and chickens, inducing obesity in the animals. The present study aimed to confirm whether companion animals, like dogs, can also host HAdV-D36 and which biochemical markers can be helpful to indicate its presence. The blood of 118 canine subjects was analysed using serological techniques for determining the presence of antibodies and screened for biochemical parameters like leptin, cholesterol, and triglycerides. The presence of anti-HAdV-D36 antibodies was confirmed in 80 of the 118 tested individuals. The investigation of the relationship between antibody levels and the concentrations of cholesterol, triglycerides, and leptin in blood serum revealed a positive correlation between antibody titers and triglyceride levels in the tested samples. Furthermore, utilizing cell cultures facilitated successful isolation of HAdV-D36 from the adipose tissue of 14 dogs under investigation. Our study adds to general knowledge of the range of hosts susceptible to the pathogen in question and highlights the discrepancies in the virus mechanisms found in the existing literature. Given the diverse host range of the virus, which extends to companion animals, a zoonotic potential cannot be ruled out, which, however, requires further extensive research.