Abstract : Bacillus spp. has a diverse set of properties that allow them to thrive in a variety of natural settings. Ultrasound illustrated a significant effect on the viability of bacterial form wounds swabs. One hundred soil samples were collected from Baghdad in order to isolate B. subtitles. Also, forty clinical samples were collected from wounds swabs collected from different hospitals in Baghdad, Bacterial isolates were identified used standard techniques and diagnosed using VITEK® 2 system. Ammonium sulfate salt method were employed to extracted the crude Subtilosin in a saturation rate ranging from 40-80 %, then the mixture concentrated using cooled centrifugation at speed of 6000 rpm for 30 minutes at a temperature 4°C and the sediment was collected. Gel electrophoresis using Sephadex G-150 was prepared/ Sample obtained from the previous step was applied onto the column. Elution was achieved at a flow rate of 30 mL/hr and the same buffer was used for equilibration. Absorbance of each fraction was measured at 280 nm. Enzyme activity was also determined in each fraction and protein concentration was determined according to standard techniques. Congo red and micro-titter method employed to detect the bacterial ability to produce the biofilm. Assessment of the effect of subtilosin A on the biofilm activity was detected via one ml of synthesized subtilosin of different concentration (100, 50, 25 and 12.5 mg ̸ ml) was added to 9 ml of Congo agar medium then left to dry at room temperature, plates were inoculated with pathogenic isolates and incubated at 37°C for 24 to 48 h. The micro-titer plate anti-biofilm assay estimates the percentage of bacterial biofilm reduction in relation to the control wells, which were set at 100% to indicate the absence of subtilosin. Bacterial suspensions (200 ml) were placed in a reaction vessel (250ml beaker) and sonicated by placing the reaction vessel containing the bacterial suspension (beaker) into the water of a 40 kHz ultrasonic cleaning bath (Langford Sonomatic 3757T Sonomatic Ltd. Birmingham, England). The temperature was maintained at 25 oC by employing ice bags. Sonication was performed at Power setting 100% intensity). Initial experiments analyzed using colony forming units (CFU/ ml) and colormtic effect. Enumeration of bacteria following experiments analyzed using Serial dilution and standard plate counts Results were converted into CFU/ml. Combined effect of ultrasound (40kHz, intensity 100%) and subtilosin (100, 50, 25 and 12.5%) together on the biofilm of different bacterial isolates. Samples of 200ml of Bacterial suspensions treated with ultrasound (40kHz, intensity 100%) for 30 minutes. Samples were taken after 0,5,10,15 and 30 minutes, then treated bacterial samples incubated with subtilosin (100, 50, 25 and 12.5%) incubated at 37°C for 24 to 48 h, Samples were analysed using spectrophotometry to measure the OD. Forty swab were collected from wounds taken from different Baghdad's hospitals, bacterial isolates showed that highest incidence of bacteria were Pseudomonas (35%) followed by 17.5% K. pneumoniae, 15 % E. coli, 10 % each of S. aureus and A. baumannii, Proteus 7.5 % and Morganella 5%. Effect of subtilosin 100% on the biofilm production alone were detected using well diffusion methods to measure the Inhibition zone (IZ) for the subtilosin on bacterial isolates, result revealed that Acinetobacter baumannii significant inhibition 6 mm, followed by E.coli with 5mm, then Pseudomonas aeruginosa with only 3 mm I.Z. On the other hand, Staph. auras demonstrated a highly resistance for subtilosin with only 2 mm of I.Z. combined treatment of ultrasound 40 KHz bath and subtilosin (100%) causing a notably increase in the efficiency of Subtilosin while exposing the bacterial isolates to an ultrasound bath for 30 minutes only. The aims of current research is isolation and purification of subtilosin from B. subtilis bacteria and evaluation of subtilosin anti biofilm activities separately, as well as, assessment the effect of sonication (40 kHz) bath as combination treatment to identify a synergistic effect on the biofilm.