Phytases are enzymes of great industrial importance with wide range of applications in animal and human nutrition. These catalyze the hydrolysis of phosphomonoester bonds in phytate, thereby releasing lower forms of myo-inositol phosphates and inorganic phosphate. Addition of phytase to plant-based foods can improve its nutritional value and increase mineral bioavailability by decreasing nutritional effect of phytate. In the present investigation, 43 phytase positive bacteria on PSM plates were isolated from different sources and characterized for phytase activity. On the basis of phytase activity and zone of hydrolysis, two bacterial isolates (PSB-15 and PSB-45) were selected for further characterization studies, i.e., pH and temperature optima and stability, kinetic properties and effect of modulators. The phytases from both isolates were optimally active at the pH value from 3 to 8 and in the temperature range of 50–70 °C. Further, the stability of isolates was good in the pH range of 3.0–8.0. Much variation was observed in temperature and storage stability, responses of phytases to metal ions and modulators. The K m and V max values for PSB-15 phytase were 0.48 mM and 0.157 μM/min, while for PSB-45 these were 1.25 mM and 0.140 μM/min, respectively. Based on 16S rDNA gene sequence, the isolates were identified as Serratia sp. PSB-15 (GenBank Accession No. KR133277) and Enterobacter cloacae strain PSB-45 (GenBank Accession No. KR133282). The novel phytases from these isolates have multiple characteristics of high thermostability and good phytase activity at desirable range of pH and temperature for their efficient use in food and feed to facilitate hydrolysis of phytate-metal ion complex and in turn, increased bioavailability of important metal ions to monogastric animals.