Biosurfactants are much-discussed alternatives to petro- and oleochemical surfactants. Alcanivorax borkumensis, a marine, Gram-negative γ-proteobacterium, produces a glycine-glucolipid biosurfactant from hydrocarbons, pyruvate, and acetate as carbon sources. Sustainable acetate production from lignocellulose or syngas adds to its relevance for the bioeconomy. This study investigated nitrogen sources and carbon-to-nitrogen ratios (C/N) to optimize fed-batch fermentation for biosurfactant production using A. borkumensis with acetate as the carbon source. Urea enabled high biosurfactant production, which was confirmed in DO-based fed-batch fermentation. Varying C/N ratios led to increased glycine-glucolipid production and decreased biomass production, with improvement plateauing at a C/N ratio of 26.7 Cmol Nmol−1. pH-stat fed-batch fermentation using glacial acetic acid as the carbon source and a pH-adjusting agent doubled the biosurfactant production. Finally, bubble-free membrane aeration was used to prevent extensive foam formation observed during conventional bubble aeration. The efficient production made it possible to investigate the bioactivity of glycine-glucolipid in combination with antibiotics against various microorganisms. Our findings allow for the leverage of glycine-glucolipid biosurfactant production using acetate as a carbon source.