Andrimid, a known non-ribosomal pseudo-peptide antibiotic, was isolated from a psychrotolerant Serratia proteamaculans strain. The antibiotic peptide was produced at low temperature (8 °C) in a 7.5 l BIOFLO 101 bioreactor under batch culture mode. Andrimid activity from S. proteamaculans culture was only detected at 25 °C and below and potent antibacterial activity was revealed against both, pathogenic and non-pathogenic bacteria. Minimal inhibitory concentration values determined by microdilution experiments varied in the range between 0.01 and 0.78 μg/ml. Antimicrobial purification and structure elucidation were carried out by LC-MS/MS and ¹H/¹³C NMR approaches. The effects on the ultrastructure of sensitive Escherichia coli 35,218 cells were observed by transmission electron microscopy at different inhibition stages. This work demonstrated the significance of bioprospection from cold environments through the screening of microorganisms with ability to produce cold-active biomolecules of biotechnological interest. S. proteamaculans 136 was revealed as a novel microbial source for andrimid production at low temperatures, showing biotechnological potential to be applied in cryopreservation, food or cosmetic industries against pathogenic bacteria.