Streptococcus pneumoniae is one of the major general causes of important diseases world-wide such as pneumonia and meningitis with a high morbidity and mortality in adults especially in elderly people. Pneumococcal biofilms are essential for colonization and persistence, and it is important to continue to investigate the environmental conditions which affect their formation. This study came to find out the adaptability of biofilm formation in S. pneumoniae that causes pneumonia in Diyala city and diagnose them with PCR method by using 16SrRNA for pneumococcal detection in addition to the roads bacteriology culture test. In this study, 192 sputum samples were collected from Bacteriology Unit of Baquba teaching hospital, Chest Diseases Consultation Clinic and Al-Khalis General Hospital. It appeared that 122 (64%) samples showed no growth while 70 (36%) showed positive growth. Streptococcus pneumoniae isolates were obtained from 32 isolates. The results of this study showed amplify 1500 bp domain of 16SrRNA by PCR, which was positive in 32 (100%). In addition, the PCR product of the samples were sent for sequencing to identify the Streptococcus pneumoniae strains. The results appeared that the strains of S. pneumoniae that cause pneumonia in Iraq especially in Diyala city are K10 and D39 strains. Biofilm formation assay was measured by using microtiter plate in three different media and the membrane formation was measured at four times of bacterial growth phases to show the adaptability of biofilm formation in different environmental conditions in different media culture. Data of the current study showed that the ability of pneumococcal to form biofilm was better when using a poor medium such as nutrient broth, and the highest value was at the stationary phase.