The factors regulating the assembly of the three-dimensional structure of Streptococcus mutans biofilms remain obscure. Polyamines are essential in biofilm formation of certain bacteria. Norspermidine, an unusual polyamine, has been a controversial polyamine that can lead to biofilm disassembly. However, the role of norspermidine in S. mutans biofilms remains unknown. Therefore, the present study investigated the impact of norspermidine on S. mutans biofilms. The different architectures of the biofilms in norspermidine and control groups indicated that the basic units, bacteria‑exopolysaccharide units (BEUs), represent the exopolysaccharide (EPS) and bacterial assembly pattern in S. mutans biofilms. In addition, norspermidine inhibited S. mutans biofilm formation and changed the basic composition of the biofilm, which led to an unusual EPS architecture. Therefore, 5 mM norspermidine inhibited biofilm formation both by decreasing the rate of cell viability and changing the biofilm structure. Gene‑expression microarray analysis indicated that the formation of an irregular architecture in the norspermidine group was potentially attributable to the downregulation of elements of the quorum‑sensing system (by 2.7‑15‑fold). The present study suggested that the BEUs are a basic structure of S. mutans biofilm and its assembly is regulated majorly by the quorum‑sensing system. Norspermidine can lead to structure change in BEUs by influencing S. mutans quorum-sensing system.