Oxydifficidin is a natural polyketide antibiotic that is a long-known ribosome-targeting antibiotic that inhibits protein synthesis. In this paper, we describe Bacillus velezensis strain EV17 and compare its complete genome sequence with that of the previously characterized strain K-3618 and the difficidin biosynthetic gene cluster (BGC) combined with mass spectrometry, to elucidate the production of oxydifficidin by these strains. Isolated oxydifficidin was determined to increase generalized inhibition of translation at each step of protein biosynthesis using toeprinting and small fluorescent peptide assays. In previous studies, it has been demonstrated that oxydifficidin targets L7/L12 protein. Although spontaneous mutations in ribosomal protein L7/L12, located relatively close to the thiostrepton binding site on uL11, confer resistance to oxydifficidin, our data show that oxydifficidin binding does not interfere with thiostrepton, thereby refining previous findings about its putative ribosomal target. We show that this compound does not affect eukaryotic translation and has no toxic effect on eukaryotic cells. These facts are important to further investigate its potential as a bioprotectant against phytopathogens or even as a therapeutic activity.