In this research work, Random amplified polymorphic DNA (RAPD) markers Protocol was used to characterize bacterial isolates from two selected General Hospitals Waste Water (HWW). The hospital environment and its wastes accumulate diseases from both inward and outward patients. It is pertinent to investigate the wastewater and study its microbial community. Wastewater samples were collected from two major General Hospitals in Akoko area, namely, Ikare and Akungba-Akoko General Hospitals. Samples were microbiologically examined for the presence of bacterial colonies. Isolated bacterial species were preliminarily identified through conventional biochemical tests and were further characterized using 16s rRNA sequencing protocol. The bacterial gene was amplified using selected primers. DNA was isolated, purified, and amplified using 16s rRNA gene sequence protocol, and the results were analyzed and assessed with the standard bacteria sequence in the NCBI database. Molecular evolutionary analyses and Phylogenetic trees were mapped out from the results obtained. it was observed that the Bacterial counts in Cfu/ml range between 10.0 x 10³Cfu/ml to 24.0 x 10³Cfu/ml. The highest bacterial count was observed from the test sample from Ikare followed by Akungba Akoko General Hospitals with the Cfu/ml value of 24.0 x 10³ and 18.0 x10³ Cfu/ml respectively. Molecular data sequence, when compared with NCBI database using BLAST showed 99.60 –99.87% bactaria phylogenic identity and E-value equal to 0 for all closely related taxa. The following bacteria were characterized using Random amplified polymorphic DNA (RAPD) markers protocol namely: Streptococcus pneumoniae, Staphylococcus aureus, Bacillus cereus, and Bacillus subtilis. this study has revealed the presence of different bacterial species in hospital waste water which pose threat to public health. Hence, proper monitoring and sewage treatment should be encouraged in different hospital settings before the disposal of these wastewaters to the public water runways