The family Actinomycetaceae comprises several important pathogens that impose serious threat to human health and cause substantial infections of economically important animals. However, the phylogeny and evolutionary dynamic of this family are poorly characterized. Here, we provide detailed description of the genome characteristics of Trueperella pyogenes, a prevalent opportunistic bacterium that belongs to the family Actinomycetaceae, and the results of comparative genomics analyses suggested that T. pyogenes was a more versatile pathogen than Arcanobacterium haemolyticum in adapting various environments. We then performed phylogenetic analyses at the genomic level and showed that, on the whole, the established members of the family Actinomycetaceae were clearly separated with high bootstrap values but confused with the dominant genus Actinomyces, because the species of genus Actinomyces were divided into three main groups with different G+C content. Although T. pyogenes and A. haemolyticum were found to share the same branch as previously determined, our results of single nucleotide polymorphism tree and genome clustering as well as predicted intercellular metabolic analyses provide evidence that they are phylogenetic neighbors. Finally, we found that the gene gain/loss events occurring in each species may play an important role during the evolution of Actinomycetaceae from free-living to a specific lifestyle.