In this study, different N-alkyl-beta-D-glucosylamines were evaluated for both antifungal and antibacterial activity against Fusarium proliferatum (INRA, MUCL 1807.7), Listeria innocua (ISTAB, Université Bordeaux 1), and Salmonella typhimurium (Institut Pasteur 5858). The tested glucosylamines were beta-D-glucosylamine (GPA), N-ethyl-beta-D-glucosylamine (EtGPA), N-butyl-beta-D-glucosylamine (BuGPA), N-hexyl-beta-D-glucosylamine (HeGPA), N-octyl-beta-D-glucosylamine (OcGPA), N-dodecyl-beta-D-glucosylamine (DoGPA), N-(2-hydroxyethyl)-beta-D-glucosylamine (HEtGPA), N,N-di(2-hydroxyethyl)-beta-D-glucosylamine (DHEtGPA) and N,N-diethyl-beta-D-glucosylamine (DEtGPA). The effectiveness of N-alkyl length, N-substitution, and N-hydroxyalkyl groups on both antibacterial and antifungal activity were evaluated. Results indicated that these compounds exhibited different biological activities and their effectiveness was highly increased from short to long N-alkyl chains. DoGPA exhibited more potent biological activity against all target strains than other N-alkyl glucosylamines tested. Using a radial growth method, we demonstrated that this compound completely inhibited fungal growth at 0.5 x 10(-4) mol mL(-1), while OcGPA and HeGPA lead to 71% and 43% fungal inhibition, respectively. Using the coating method, we demonstrated that DoGPA completely inhibited bacterial growth at 0.025 x 10(-4) and 0.05 x 10(-4) mol mL(-1) for L. innocua and S. typhimurium, respectively, while at the same concentrations, OcGPA exhibited weaker antibacterial activity of 12% and 27%, respectively, for L. innocua and S. typhimurium. The hole plate method enabled us to estimate the minimum inhibitory concentration (MIC) of DoGPA found to be 0.02 x 10(-4) and 0.025 x 10(-4) mol mL(-1) for L. innocua and S. typhimurium, respectively. Glucosylamines with N-hydroxyalkyl and short N-alkyl chains varying from C-2 to C-4 exhibited weaker antimicrobial activity.