Aquaculture faces demanding challenges in the foreseeable future, especially the necessity to prevent and control infectious diseases outbreaks and the overuse of antibiotics, with the consequent emergence of antibiotic-resistant bacteria. Therefore, there is a serious urgence to develop alternative antimicrobial strategies to antibiotherapy. In this scenario, ribosomally synthesized antimicrobial peptides, i. e. bacteriocins, exert a high antimicrobial potency and low toxicity, not affecting gut commensal microbiota. Nisin Z (NisZ), a natural variant of nisin A (NisA), is a 34-residue-long lantibiotic bacteriocin, produced by several Lactococcus lactis strains, showing a remarkable inhibitory spectrum against several bacterial ichthyopathogens. In this work, two different multi-chromatographic procedures were used to purify NisZ from cell-free culture supernatants from Lactococcus lactis RBT18, isolated from cultured rainbow trout. In both cases, MALDI-TOF MS analyses of the peptides confirmed the presence of NisZ (3,330 Da), and the yields and antimicrobial activity results were compared. The first purification procedure, a modification of the protocol described by Cintas et al. (1995), resulted in an absorbance peak (eluted at 58% elution buffer, v/v) showing antimicrobial activity, with a yield of 77% and a 348,000-fold increase in the specific antimicrobial activity compared to that of the cell-free supernatant. The second purification procedure, based on the protocol described by Field et al. (2012), resulted in two antimicrobial active absorbance peaks (55 and 58% elution buffer, v/v, respectively), with a combined yield of 4% and a 16,000-fold-increase in the initial specific antimicrobial activity. In summary, our results show that while both procedures are suitable for NisZ purification the former is more appropriate and effective. Nevertheless, further research is necessary to increase production and optimize purification, to obtain economically viable yields that allow its use as an alternative to antibiotics in aquaculture.