The maintenance of genomic stability is critical for the proper functioning of all organisms. Various DNA repair processes are involved in safeguarding the integrity of the genome, including homologous recombination (HR), a process that repairs double-stranded breaks in the DNA. The DNA motor protein Rad54 is a highly conserved DNA translocase that is crucial for HR function. In the human context, mutations in its homolog, Rad54L, are known to be associated with many cancer types. This study utilized the model system of Saccharomyces cerevisiae to examine the effects of mutations in Rad54 on important cellular phenotypes. rad54R272Q and rad54R272A demonstrated significant growth defects in response to DNA damage, exhibiting a gradient of activity when compared to the WT. Intriguingly, diploid rad54R272A/rad54R272A displayed a growth defect even under normal conditions. Additionally, the study demonstrated no significant difference in template switching frequencies between the WT and mutants. Together, these results provide greater insights into Rad54’s function in HR and illuminate how mutations within this protein can lead to genome instability, which can contribute to cancer development in humans.