In the absence of information on Borrelia species causing Lyme borreliosis (LB) in Serbia, we were interested in the pathogenic potential of local strains of Borrelia burgdorferi sensu lato isolated from ticks. The susceptibility of B. burgdorferi sensu lato isolates to human complement was tested in vitro. A total of 31 strains were analysed by serum susceptibility testing - 12 Borrelia afzelii, 12 Borrelia lusitaniae, three Borrelia bavariensis, two Borrelia garinii, and two Borrelia valaisiana. The serum-resistant strains (B. afzelii, B. bavariensis and B. valaisiana) expressing OspA and OspB or OspA and OspC proteins might be related to the different pathogenic potential of the specific Borrelia strains and their ability to cause different clinical manifestations of LB in humans. Among the B. lusitaniae strains from Serbia, two were significantly more motile than others (all p < 0.05) and equally motile as human isolate (both p > 0.05), and since they express OspC, their pathogenic potential remains unclear. For further studies, it is of particular interest to explore the mechanism of resistance of B. valaisiana and to explain the fair motility of B. lusitaniae, a predominant Borrelia species in Serbia, in the presence of human complement.