Background: The fermentation substrate efficiency of glucose supplemented with guava seed flour (GGSF) or glucose supplemented with dry Aspergillus niger mycelium (GANM) was evaluated during the production of biomass and antimicrobial compounds by the lactic acid bacteria Weissella confusa. Results: The fermentation substrate efficiency was measured by comparing the biomass formation, substrate consumption, substrate conversion, antimicrobial activity and product yield. The antimicrobial activity was measured against a commercial Staphylococcus aureus strain. The results were compared against fermentations performed in a commercial substrate (CS), the MRS (Man-Rogosa-Sharpe) substrate. The fermentations were performed discontinuously for 4 hrs at 100 rpm and 32ºC. The biomass production exhibited a statistically significant difference (P ≤ 0.05) between treatments. The biomass production was 13.98% higher in the CS than in the GGSF and GANM substrates; however, there were no statistically significant differences for the specific growth rate. Conclusions: The GGSF and GANM substrates favored an antimicrobial activity against Staphylococcus aureus during the second and third hours of fermentation (inhibition diameter was 6.11% and 4.72%, respectively). The GGSF, GANM and CS substrates did not present statistically significant differences for the production of antimicrobial substances against Staphylococcus aureus. Therefore, GGSF and GANM can be considered as viable and economical alternative nitrogen sources for the production of the antimicrobial compounds formed by Weissella confusa in submerged fermentations.