The main objective of the present study was to desalinate seawater using Bacillus cereus gravel biofilm and cellulose acetate (CA) membranes with and without silver nanoparticles (AgNPs) as a potent and safe disinfectant for the treated water. Six desalination trials (I, II, III, IV, V and VI) were performed using the proposed biofilm/cellulose membrane. Results confirmed that Bacillus cereus gravel biofilm (microbial desalination) is the optimal system for desalination of seawater. It could achieve 45.0% RE (initial salinity: 44,478 mg/L), after only 3 h compared to the other tested treatments. It could also achieve 42, 42, 57, 43 and 59% RE for TDS, EC, TSS, COD and BOD, respectively. To overcome the problem of the residual salinity and reach complete elimination of salt content for potential reuse, multiple units of the proposed biofilm can be used in sequence. As a general conclusion, the Bacillus cereus biofilm system can be considered as remarkably efficient, feasible, rapid, clean, renewable, durable, environmentally friendly and easily applied technology compared to the very costly and complicated common desalination technologies. Up to our knowledge, this is the first time microbial biofilm was developed and used as an effective system for seawater desalination.