The in vitro cytotoxicity of the antimicrobial peptide P34 was evaluated in different eukaryotic cells. The food-grade bacteriocin nisin was also analysed for comparison. Vero cells were treated with different concentrations (0.02–2.5 μg·ml−1) of antimicrobial peptide P34 and nisin. Cell viability and plasma membrane integrity were checked by MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide], NRU (Neutral Red dye uptake) and LDH (lactate dehydrogenase) assays. The EC50 values of the peptide P34 in MTT and NRU assays were 0.60 and 1.25 μg·ml−1 respectively, while values of nisin found were 0.50 and 1.04 μg·ml−1. In the LDH assay, the EC50 values were 0.65 and 0.62 μg·ml−1 for P34 and nisin, respectively. The peptide P34 revealed similar haemolytic activity on human erythrocytes (5.8%) when compared with nisin (4.9%). The effects on viability, motility and acrosomal exocytosis of human sperm were also evaluated. Nisin and P34 showed similar effects on sperm parameters. The evaluation of cytotoxicity of antimicrobial peptides is a critical step to guarantee their safe use.