The production of antimicrobial metabolites by Paenibacillus polymyxa RNC-D was assessed. Two process variables, glucose and inoculum concentrations, were evaluated at different levels (5-40 g L -1 , and at ϕr = 2.5-5.0 %, respectively), and their effects on biomass formation, minimal inhibitory concentration (MIC) against Escherichia coli, and surface tension reduction (STR) were studied. When the fermentation process was carried out under non-optimised conditions, the biomass, MIC, and STR achieved the following values: 0.6 g L -1 , 1 g L -1 , and 18.4 mN m -1 , respectively. The optimum glucose (16 g L -1 ) and inoculum volume ratio (ϕr = 5.0 %) were defined in order to maximise the biomass formation, with a low value of MIC and high STR of extract. The experiments carried out under optimal conditions showed the following values for the dependent variables: biomass concentration 2.05 g L -1 , MIC 31.2 µg mL -1 , and STR 10.7 mN m -1 , which represented improvement of 241.7 %, 96.9 %, and 41.9 % for the responses of biomass, MIC, and STR, respectively. This is the first recorded study on the optimisation of culture conditions for the production of antimicrobial metabolites of P. polymyxa RNC-D, and constitutes an important step in the development of strategies to modulate the production of antimicrobial molecules by this microorganism at elevated levels.