Microbial culture collections are important resources for isolation of natural compounds with novel properties. In this study, a culture collection of around 1,500 pigmented heterotrophic bacteria was established. The bacteria were isolated from the sea surface microlayer at different sampling sites along the mid-part of the Norwegian coast. The bacterial isolates produced pigments of various coloration (e.g. golden, yellow, red, pink and orange). Methanol extracts of sixteen isolates were characterized with LC-Diodearray-TOF mass spectrometry analysis. The number of pigments per isolate varied considerably, and a tentative identification of the pigments was performed based on UV-absorbance profile and molecular formula assignation based on the accurate mass determination. The LC-MS analyses revealed that most of the pigments probably were carotenoids. Furthermore, we developed a high throughput LC-MS method for characterization and screening of a larger sub-fraction (300 isolates) of the culture collection. The aim was to screen and identify bacterial isolates producing carotenoids that absorb light in the UVA-Blue light. Six of the bacterial strains were selected for detailed investigation, including 16s rRNA sequencing, preparative HPLC for purification of major carotenoids and subsequent structural elucidation with NMR. Among the identified carotenoids were zeaxanthin, nostoxanthin and sarcinaxanthin, some with novel glycosylation patterns.