One of the most important problems to the fish in aquaculture is pathogenic bacteria. Therefore is a serious necessity in aquaculture to upgrade microbial hegemony strategies. The present study focused on isolation, screening, biochemical and molecular level characterizations of potential probiotic bacteria from the fish pond in Basra city southern Iraq. Isolated bacteria were characterized based on their morphological and biochemical traits and were identified using automated instrument (Vitek II) and 16S rRNA gene sequencing. As a result, bacteria were identified as Pediococcus pentosaceus. In order to be used in aquaculture as probiotics, bacteria must have good probiotic properties such as the ability to live in the presence of bile salts and low pH. Hence, bacteria were incubated with different concentrations of bile salts and pH values for different periods of time. In addition to that, bacteria were subjected to additional tests, such as tolerance to survive in simulated human gastrointestinal tract conditions and antibiotic susceptibility. Results of all these tests indicated that Pediococcus pentosaceus has good probiotic properties. The pathogenic bacteria (Salmonella sp.) were identified using the automated instrument for bacterial identification (Vitek II). The antagonistic ability of P. pentosaceus toward the pathogenic bacteria (Salmonella sp.) was tested using the agar sawing method. The result indicated that P. pentosaceus has good antagonistic ability.