Both D-amino acids and the polyamine norspermidine were previously reported to trigger the disassembly of Bacillus subtilis biofilms, but two studies now question these findings. Leiman et al. confirm that D-amino acids inhibit biofilm formation, but the authors show that this is not a direct effect and that it is due to the misincorporation of these amino acids into newly synthesized proteins, which also reduces growth. Furthermore, they find that the B. subtilis strain in which D-amino acids were previously shown to trigger biofilm disassembly has a mutation in its D-aminoacyl-tRNA deacylase, which removes D-amino acids from mischarged tRNAs and thus prevents their misincorporation into proteins. Repair of this mutation abolished the inhibitory effect of D-amino acids on biofilm formation and growth. Previous work had also reported that norspermidine is produced in aged B. subtilis biofilms and that it triggers biofilm disassembly. Using high-performance liquid chromatography, Hobley et al. now find that B. subtilis biofilms do not produce detectable norspermidine, whereas it is readily detectable in cultures of Vibrio parahaemolyticus, which is a known norspermidine producer. Furthermore, the authors show that exogenously added norspermidine promotes B. subtilis biofilm formation, similarly to its structural analogue spermidine, which is a known biofilm inducer. Norspermidine inhibits both growth and biofilm formation only at very high concentrations, which are unlikely to occur naturally. Moreover, a genomic analysis showed that B. subtilis lacks the norspermidine biosynthetic pathway. In conclusion, these two studies suggest that the roles of D-amino acids and norspermidine as specific biofilm disassembly triggers are questionable and that, under certain conditions, these substances are general growth inhibitors that can indirectly inhibit biofilm formation.