Colletotrichum spp. and Phytophthora cinnamomi can cause severe economic losses in avocado production in postharvest as well as in crop stages. Therefore, world avocado agroindustry is demanding for new alternatives to cope with these pathogens. The main objective of this study was to assess the production and inhibitory activity of secondary metabolites from the well-known biocontrol bacterium Serratia marcescens against avocado pathogens. S. marcescens strain ARP5.1 was obtained from the rhizoplane of an apparently healthy avocado tree. A bioactivity-guided methodology was used to isolate molecules of interest. Minimal medium standardization was performed in order to increase the overall activity of the crude extract. One factor at a time approach and a Plackett-Burman (PB) design were carried out for the selection of appropriate carbon and nitrogen sources and the most influencing fermentation parameter among nine variables. As a result, three active compounds (prodigiosin, serratamolide and haterumalide NA) were isolated from the crude extract and structurally elucidated. Haterumalide NA proved to be the most active metabolite with a MIC of 5 and < 0.16 µg mL-1 against Colletotrichum gloeosporioides and P. cinnamomi, respectively. Prodigiosin (6.25 and 6.25 µg mL-1) and serratamolide (80 and 80 µg mL-1) were less active. Highest production of haterumalide NA was achieved in culture media consisting of maltose and yeast extract and PB experiments revealed maltose concentration, pH and Mg2+ as the most influencing factors for haterumalide NA production (p < 0.05). With these optimisations the production rate could be increased 16.7 fold compared with the basic minimal medium composition. Our results show that haterumalide NA is a highly effective compound for the control of avocado diseases and a promising candidate to become a commercially available product due to its easy production and strong antioomycete activity.