Nisin is the prototypical example of the lantibiotic family of antimicrobial peptides and has been employed as a food preservative for over half a century. It has also attracted attention due to its potency against a number of multidrug-resistant clinical pathogens. Nisin A is the originally isolated form of Nisin and a further five natural variants have been described which differ by up to 10 amino acids (of 34 in total in Nisin A). Nisins A, Z, F and Q are produced by Lactococcus lactis, while Nisins U and U2 are produced by Streptococcus sp. In this study we bioengineered the nisA gene of a Nisin A producer to generate genes encoding Nisins Z, F, Q, U and U2. We determined that while active Nisin Z, F and Q can be produced against this genetic background, active forms of Nisin U and U2 are not generated. Minimum inhibitory concentration studies with Nisin A, Z, F and Q variants against a series of different clinically significant pathogens establish differences in specific activities against selected targets. Nisin F was most impressive, being the most active, or one of the most active, against the MRSA strain ST 525, EC 676, EC 725, VISA 22900, VISA 22781, hVISA 35197, Staphylococcus aureus 8325-4 and L. lactis HP. Nisin Z was most active against ST 299, hVISA 32683 and, together with Nisin F, HP but had contrastingly poor activity against ST 525, EC 676 and 8325-4. Nisin F, Q and A exhibited similar potency against VISA 22900. This was the only target against which Nisin Q and Nisin A were among the most active variants.