Toxin-antitoxin (TA) systems are small genetic elements of prokaryotes associated with persister cell formation, phage defence, stress regulation and programmed cell arrest. In this study, we characterized two paralogues of the ribosome-dependent RNase YefM-YoeB TA system from the Gram-positive organism Staphylococcus equorum SE3. 5' Rapid amplification of cDNA ends confirmed transcriptional activity in the exponential growth phase and revealed an extended 5' untranslated region upstream of the yefM-seq1 gene. Inducible expression of the putative yoeB-seq1/2 toxins led to growth defects of Escherichia coli, which were counteracted by simultaneous induction of the cognate yefM-seq1/2 antitoxin candidates in a strictly pairwise manner. Bacterial two-hybrid assays revealed interaction between YoeB-seq1 and YefM-seq1 but not YoeB-seq1 and YefM-seq2, also indicating two independent systems. In vivo primer extensions demonstrated specific RNA cleavage adjacent to the start codons by YoeB-seq proteins, and YoeB-seq2 activity could be neutralized by the corresponding antitoxin YefM-seq2. Together, these results indicate that the two yefM-yoeB-seq1/2 paralogues from S. equorum encode functional TA systems.