Among many sources of natural bioactive substances, mushrooms constitute a huge and mostly unexplored group. Biologically active secondary metabolites of Ganoderma, a group of wood-degrading mushrooms, have recently been reviewed. Our previous study revealed the antimicrobial activity of extracts from G. resinaceum grown in submerged culture against phytopathogens. Different factors can influence the production of secondary metabolites, including nutritional factors. In this study we evaluated the influence of different culture conditions on the antimicrobial activity of extracts from liquid cultures of G. resinaceum, through use of a factorial design. Minimum inhibitory concentrations for extracts produced under different culture conditions were determined against Staphylococcus aureus and Xanthomonas vesicatoria. Based on the results of these assays, larger-scale cultures in malt extract broth supplemented with 20 g/L glucose and a 15-day incubation time should be performed in order to isolate from G. resinaceum antibiotic compound(s) that are potentially useful against S. aureus. In addition, pH 5 should be considered for the production of antimicrobial metabolites against X. vesicatoria from supernatant broths or extracts from G. resinaceum.