Abstract Bacteriophages are able to hijack host essential machineries to benefit their fitness and assemble their own progeny. Phage proteins targeting major bacterial pathways can be powerful tools to understand cell functions and have possible applications in human health and industry. Bacterial genomes also harbor cryptic prophages carrying genes that may contribute to their host fitness and properties. The cryptic prophages are mostly transcriptionally silent and most of the functions they encode are not annotated. In B. subtilis , the 48 kb-long skin element is a prophage carrying the yqaF-yqaN operon, which is tightly regulated by the Xre-like repressor sknR . The small yqaH gene potentially encodes the protein YqaH in absence of SknR. It was previously reported that YqaH interacts with the replication initiator DnaA in yeast two-hybrid assay and its expression in B. subtilis causes defects in the chromosomal cycle. In this study, we report that, in addition to DnaA, YqaH interacts with Spo0A, a master regulator of sporulation. To decipher yqaH mode of action, we used the yeast two-hybrid to isolate single mutations in yqaH that separate interactions with DnaA and Spo0A. We isolated mutations that caused loss-of-interaction (LOI) with DnaA but not Spo0A. However, all mutations disrupting the interaction with Spo0A were also DnaA-LOI functions, suggesting that these functions could not be separated. We found that expression YqaH carrying DnaA-LOI mutations affects both chromosome integrity and DnaA-mediated transcription, leading to growth inhibition as well as preventing bacterial development such as sporulation and biofilm formation. These results show that YqaH acts as an antimicrobial peptide in B. subtilis and pave the way for the structural design of mutants with improved antibacterial action.