Background & Aims of the Study: Rhodococci according to possess large genome, their active metabolism and survival under extreme conditions, are highly regarded for biodegradation and bioremediation of different pollutants especially heavy metals in the environment. Biofilms are more resistant to heavy metals than free-swimming organisms. The aim of this study was biofilm formation of two native Rhodococcus strains (Rhodococcus rhodochrous and Rhodococcus rhodnii) and comparative evaluation effects of heavy metals such as lead, copper, zinc, chromium and cadmium in various concentrations against biofilm and planktonic cells that carried out at the first time. Materials & Methods: Bacterial strains used in this investigation were isolated from agricultural soils in Qom, Iran. Biofilm formation was performed at 30 °C and 37 °C in tryptic soy broth (TSB) and brain heart infusion (BHI) broth, at 24,48,72 and 96 hours in 96-well microplate. Minimum inhibitory concentration (MIC) of heavy metals in various concentrations for planktonic cells was determined, using microdilution and macrodilution methods. Minimum biofilm inhibitory concentration (MBIC) and Minimum biofilm eradicating concentration (MBEC) in various concentrations of heavy metals for biofilm cells were evaluated by microdilution method. The result of this study confirmed, using scanning electron microscope (SEM). Results: The best condition for biofilm formation of native R.rhodochrous isolate was determined after incubation at 37°C in BHI broth, at 96 hours and the best condition for biofilm formation of native R.rhodnii isolate was determined at 30°C in BHI broth, at 96 hours. MIC of heavy metals for planktonic cells of isolates for cadmium, zinc and lead was 8 mM and assayed for copper and chromium respectively 4 and 1 mM. MBEC of heavy metals for biofilm cells of isolates for cadmium, zinc and lead was 16 mM and assayed for copper and chromium respectively 8 and 4 mM. The results of present study showed biofilm cells of native Rhodococcus isolates were 2 times more resistance to lead, copper, zinc and cadmium than planktonic cells while biofilm cells were 4 times more resistance than planktonic cells to chromium. Conclusions: Biofilm formation of R.rhodochrous and R.rhodnii and their high resistance to various concentrations of heavy metals especially cadmium show that inoculation of these native Rhodococcus isolates to contaminated agricultural soils with heavy metals, have an effective role for bioremediation.