Bacteriophages (phages), viruses that infect bacteria, are considered to be highly host-specific. To add to the knowledge about the evolution and development of bacteriophage speciation toward its host, we conducted a 21-day experiment with the broad host-range bacteriophage Aquamicrobium phage P14. We incubated the phage, which was previously isolated and enriched with the Alphaproteobacteria Aquamicrobium H14, with the Betaproteobacteria Alcaligenaceae H5. During the experiment, we observed an increase in the phage's predation efficacy towards Alcaligenaceae H5. Furthermore, genome analysis and the comparison of the bacteriophage's whole genome indicated that rather than being scattered evenly along the genome, mutations occur in specific regions. In total, 67% of the mutations with a frequency higher than 30% were located in genes that encode tail proteins, which are essential for host recognition and attachment. As control, we incubated the phage with the Alphaproteobacteria Aquamicrobium H8. In both experiments, most of the mutations appeared in the gene encoding the tail fiber protein. However, mutations in the gene encoding the tail tubular protein B were only observed when the phage was incubated with Alcaligenaceae H5. This highlights the phage's tail as a key player in its adaptation to different hosts. We conclude that mutations in the phage's genome were mainly located in tail-related regions. Further investigation is needed to fully characterize the adaptation mechanisms of the Aquamicrobium phage P14.