Most microorganisms used biofilm formation as protection mechanism that provides cells advantages as infectivity, antibiotic resistance and survivability of a variety of infections in humans. Pseudomonas aeruginosa is an opportunistic pathogen cause of infections in cystic fibrosis patients such as pneumonia and chronic lung infections. Although biofilm control is an important in medicine, clinically, there is not available effective inhibitors of biofilm formation but some environmental factors could influence on biofilm formation. This research investigated incubation periods, temperature and iron concentration effect on the formation of biofilm which is critical for virulence by multi-drug resistant P. aeruginosa taken from the sputum of pulmonary infection patient. From these results, it was found that moderate biofilm was observed on day two and day one was low in formation of biofilm but on day three was the optimum period for P. aeruginosa to switch into a strong biofilm. We investigated the effect of temperature and found that biofilm showed the highest level at temp 37°C. After using different concentrations of iron, it was showed that biofilm production was higher at the addition of FeSO4 at 25 mM incubated at 37°C for 3 days. It concluded that some factors can affect and control the biofilm formation as a significant part of virulence system plays a critical role in antibiotic resistance in P. aeruginosa infection.