Pentamidine is a small molecule inhibitor of the Ca2+-binding protein S100B and disrupts the S100B–p53 protein–protein interaction; this is thought to restore wild-type p53 tumour suppressor function in melanoma. Additional anticancer effects may be the result of inhibition of regenerating liver family phosphatases. In this study, we have used a standardized ATP-tumour chemosensitivity assay to investigate the effect of pentamidine on cells derived from 18 skin melanoma samples and one uveal melanoma sample. The cells were tested at six concentrations from which the IC50 and IC90 were calculated. To allow comparison between samples, an indexsum was calculated based on the percentage of tumour growth inhibition at each concentration. Of the skin melanoma samples tested, 78% exhibited an indexsum less than 300 indicating strong inhibition. The median indexsum of 237 also indicates considerable activity against these samples. The median IC90 (30.2 μmol/l) may be clinically achievable in a proportion of patients. The uveal melanoma sample exhibited an indexsum of 333 indicating moderate inhibition, and 86% inhibition at test drug concentration (37.96 μmol/l). These results show that pentamidine has activity against melanoma, and support the prospect of its development for therapeutic use.