Aims: To investigate the effects of growth conditions related to marine habitat on antibiotic production in sponge-derived Salinispora actinobacteria. Methods and Results: Media with varying salt concentration were used to investigate the effects of salinity in relation to Salinispora growth and rifamycin production. The chemotypic profiles of the model strain Salinispora arenicola M413 was then assessed using metabolomic fingerprints from high-pressure liquid chromatography with diode array detection (HPLC-DAD) and multivariate data analysis, before extending this approach to two other strains of S. arenicola. Fingerprint data were generated from extracts of S. arenicola broth cultures grown in media of varying salt (NaCl) concentrations. These fingerprints were then compared using multivariate analysis methods such as principal components analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA). From the analysis, a low-sodium growth condition (1% NaCl) was found to delay the onset of growth of the model S. arenicola M413 strain when compared to growth in media with either 3% artificial sea salt or 3% NaCl. However, low-sodium growth conditions also increased cell mass yield and contributed to at least a significant twofold increase in rifamycin yield when compared to growth in 3% artificial sea salt and 3% NaCl. Conclusions: The integration of HPLC-DAD and multivariate analysis proved to be an effective method of assessing chemotypic variations in Salinispora grown in different salt conditions, with clear differences between strain-related chemotypes apparent due to varying salt concentrations. Significance and Impact of the Study: The observed variation in S. arenicola chemotypic profiles further suggests diversity in secondary metabolites in this actinomycete in response to changes in the salinity of its environment.