Aims The aim of this research was production of polyphosphate (poly P) and study on its antibacterial effects. Methods and Results Poly P granules in the cells were observed with the help of Albert staining and extracted by Mussig-Zufika method. Thin layer chromatography and nuclear magnetic resonance spectroscopy (31P NMR) were used to characterize properties of these granules. Relation of phosphorus consumption and poly P production with growth was determined by the vanado-molybdate colorimetric method. Among the 60 strains of bacteria isolated from the environmental samples, strain G11 showed ability for the formation of high levels of poly P. Phylogenetic analysis showed that this isolate had 98% similarity with Bacillus megaterium. 16S rRNA sequence of isolate was deposited in GenBank with accession number JX115009. The average poly P chain length was 10 center dot 5 in this bacterium. The antimicrobial activity of bacterial extracted poly P was much better than chemical poly P, and its interaction with gentamicin increased the activity of this drug. The best synergistic activity of this interaction was observed for Corynebacterium glutamicum and Pseudomonas aeruginosa species. The highest adsorption of phosphorus occurred in stationary phase of growth curve, and then the amount of phosphorus increased in medium by degradation of stored poly P. Conclusions In this study, we isolated a high-level producer bacterium of poly P and extracted poly P by chemical treatment. In addition, we compared antimicrobial activity of chemical poly P with bacterial poly P and its interaction with gentamicin against both Gram-positive and Gram-negative bacteria. Significance and Impact of the Study Many studies have shown that bacteria are becoming resistant to gentamicin sulphate. In this study, we approved that Acinetobacter baumannii, a pathogenic gentamicin-resistant bacterium, is sensitive to bacterial poly P, and thus, this poly P can be substituted for gentamicin in treatment.