A preparative chromatographic isolation method involving thin-layer and liquid chromatography (LC) is described for the microbially derived antibiotic monoacetylphloroglucinol (MAPG). Isolation of MAPG along with the previously described 2,4-diacetylphloroglucinol (DAPG) confirms the production of two antimicrobial metabolites from Pseudomonas sp. strain F113. A gradient LC assay was developed for the determination of these compounds in growth culture media. A sample pretreatment procedure involving solid phase extraction on octadecylsilica is utilised prior to sample injection onto the LC column. This assay is used to monitor production of the two antibiotic metabolites from the strain of Pseudomonas. A range of nitrogen sources in the growth culture medium was tested for their ability to affect antibiotic production. A carbon source, such as galactose promotes high yields of antibiotics when the growth medium contains a nitrogen source in the form of ammonium ions, and produces low yields when the nitrogen source is added as nitrate ions. The LC assay is further applied to monitor the enzymatic acetylation of MAPG to DAPG, through an enzymatic activity named MAPG acetyltransferase which provides the final step in the elucidation of the biosynthesis of DAPG.