Vibrio tapetis, the etiological agent of Brown Ring Disease, mainly affects the Manila clam Ruditapes philippinarum. Although this bacterium is mainly known as a clam pathogen, it has been isolated from several fish species. The main aim of the present study was to further explore the variability of 27 V. tapetis isolates from bivalves and fish, considering three different aspects; in vitro virulence based on the loss of clam hemocyte adhesion properties, detection of the gene virB4 encoding for an essential component of the Type IV Secretion System, and MALDI-TOF MS characterization based on whole cell extracts. Finally, these approaches were compared and evaluated for their ability to differentiate the potential pathogenicity of the 27 isolates against the Manila clams. Among the 11 V. tapetis isolates from the common dab isolated in 2018 in Belgium, only one (2BB) showed intermediate in vitro virulence against the Manila clam, and seven carried the virB4 gene while none of the V. tapetis previously isolated from fish in 2003 showed the presence of this particular gene. Finally, the peak protein profiles generated with MALDI-TOF MS analysis from all 27 V. tapetis strains showed a clear clustering of clam pathogenic and nonpathogenic isolates suggesting that a new isolate of V. tapetis that would cluster within the clam pathogenic isolates could be potentially pathogenic to the Manila clam. Thereupon, MALDI-TOF MS typing allows rapid and cost-efficient identification of V. tapetis isolates and can be defined as a complementary method of the traditional qPCR that opens new perspectives to study the virulence of V. tapetis isolates but also to perform environmental monitoring in order to prevent outbreaks.