In the current scenario, the demand for energy is considered to be the major target for industries. Among the various sources of energies, crude oil plays a vital role. Oil recovery is achieved using conventional primary and secondary recovery methods. Inorder to recover the remaining residual oil, technologies like Enhanced Oil Recovery (EOR) are utilized which is also known as tertiary recovery. Among EOR, Microbial enhanced oil recovery (MEOR) is a technique which enables the improvement of oil recovery by injection of bio-surfactant produced by microorganisms. Bio-surfactant can retrieve unrecoverable oil from the cap rock which is held by high capillary force. Bio-surfactant is a surface active agent which can reduce the interfacial tension and reduce viscosity of oil and thereby can be recovered to the surface as the mobility of the oil is increased. Research in this area had shown promising results besides the method is eco-friendly and cost effective compared with other EOR techniques. n this current research work, in laboratory scale we produced bio-surfactant using the strain Acinetobacter Calcoaceticus (PTCC 1318) where fermentation was carried out at 40° C, pH 7.0 and 180 rpm. Bio-surfactant analytical studies revealed that Acinetobacter Calcoaceticus (PTCC 1318) has the capability to grow and produce maximum bio-surfactant leading to the reduction of interfacial tension from 51 mN/m to 8 mN/m. Further evaluation of oil recovery using sand packed column experiment gave a maximum recovery of 12% after water flooding.