Immunity to Babesia infection requires both innate and acquired responses, including cell mediated- and humoral responses. The aims of this study were to investigate the variation in selected peripheral blood lymphocyte phenotypes in dogs with virulent babesiosis at presentation and over time after treatment, and to determine whether these were correlated with the severity of clinical signs. Forty-four dogs naturally infected with B. rossi were studied and 5 healthy dogs were included as controls. Blood samples were collected from the jugular vein at admission, prior to any treatment, and at 24 h and 48–72 h. Leukocytes were incubated with canine specific, fluorochrome conjugated anti-CD3, anti-CD4, anti-CD8, and anti-B cell markers. Babesia-infected dogs were divided into complicated or uncomplicated groups on clinical grounds and in-house laboratory assays. The percentage CD3+ lymphocytes in the complicated group was lower compared to the controls (P = 0.014) and uncomplicated group (P = 0.007). The percentage CD4+ T lymphocytes in the complicated group was lower compared to the controls (P = 0.027) and uncomplicated group (P = 0.014). Both the complicated as well as the uncomplicated groups expressed a lower percentage CD8+ T lymphocytes compared to the control group (P < 0.001 and P = 0.005, respectively). The percentage B lymphocytes was higher in the complicated group at 48–72 h. These findings could indicate the presence of a functional immune suppression secondary to increased apoptosis or redistribution of effector lymphocytes and/or a combination of other immune modulatory mechanisms induced by B. rossi infection.