Vibrio alginolyticus is a common opportunistic pathogen that can cause vibriosis of marine aquatic animals. The application of phages or particularly associated protein products for the treatment of vibriosis has shown prominent advantages compared with the treatment with traditional antibiotics. In this study, the function of a holin-endolysin system from V. alginolyticus phage HH109 was characterized by examining the effect of their overexpression on Escherichia coli and V. alginolyticus. Our data revealed that the endolysin of the phage HH109 has stronger bactericidal activity than the holin, as evidenced by observing more cell death and severe structural damage of cells in the endolysin-expressing E. coli. Furthermore, the two proteins displayed the synergistic effect when the holA and lysin were co-expressed in E. coli, although no interaction between them was detected using the bacterial two-hybrid assay. Transmission electron microscopy observation revealed disruptions of cell envelopes accompanied by leakage of intracellular contents. Similarly, the bactericidal activity of the holin and endolysin against V. alginolyticus was also examined whatever the host is sensitive or resistant to phage HH109. Together, our study contributes to a better understanding of the mechanism of phage HH109 destroying the bacterial cell wall to lyse their host and may offer alternative applications potentially for vibriosis treatment.