Banana is an edible fruit produced by the genus Musa. It is an important fruit crops due to its great economic importance as well as nutritional value and high availability throughout the year. Banana plants have been growing in different climatic conditions however commercial banana plantations are primarily found in equatorial regions of the globe. Tissue culture is used for the mass production of planting material of banana. Using this technique, viral free and large numbers of clones are produced. Clonal propagation is a complex process. There are many problems in banana tissue culture. They are microbial contamination and phenolic exudation, during explant propagation, phenolics are synthesized and released in sufficient quantities under the influence of polyphenol oxidases. The present investigation aimed to identify the microbial contamination in the banana tissue culture medium which includes isolation and identification of the microbes such as fungi, bacteria etc. using conventional methods as well as molecular methods including 16S DNA study. Antibiotic sensitivity test against the bacteria to suggests better antibiotic. On identification it was found that Penicillium was the fungus present in the medium. Three types of bacteria could be isolated from the media and it could not be identified by conventional methods hence subjected for 16S DNA assay. It revealed that the bacteria present in the medium was Staphylococcus warneri (Sequence ID: MH014968.1) and the bacteria B identified as Pseudomonas nitroreducens (Sequence ID: MF627712.1). We could not identify one of the isolates using universal primers. An antibiotic sensitivity study was conducted and found that the antibiotic Tetracycline was more effective for all the bacteria isolated. However more combinations and phytotoxic studies should be conducted to ascertain its suitability.