A 2-(n-morpholino)ethane sulfonic acid (MES)-buffered minimal medium with low levels of metal chelates was used to evaluate the heavy metal (Co+2, Pb+2, Zn+2, Fe+2, Fe+3, Cd+2, Cu+2, and Cr+6) toxicity to Pseudomonas fluorescens through minimizing the limitation of the existing medium. The interaction between bacteria and heavy metals was real-time monitored by microcalorimetry and reflected by thermogenic curves. Compared with the main parameters, microbial growth rate constant k and total heat evolution Q total, a general order of toxicity was found to be Fe+3<Co+2<Fe+2<Cu+2<Cr+6<Zn+2<Pb+2<Cd+2. The main metal speciation was Co+2, Zn+2, Fe+2, Cd+2, PbOH+, Fe(OH)+2, CuOH+, HCrO4-, and CrO4(-2) at pH 6.4. These metal speciations are main factors to exhibit the metal toxicity to bacteria. To validate their interaction, the surface chemical functional groups of P. fluorescens biomass identified by Fourier transform infrared (FTIR) were amino, carboxyl, hydroxyl, and phosphoryl groups, which maybe involved in the biosorption of metal ions.