Surface water quality is an important module of the aquatic ecosystem which is crucial to wildlife and environmental health. The term water quality is sometimes extended to include biodiversity of waters and biological indicators or ecological health. Microbiological examination of water is an essential tool to investigate the presence of microorganisms that might constitute a hazard to both animal and public health. Microbiological examination of waterholes in national parks is thus crucial in assessing ecosystem health. The study therefore aimed at assessing the bacteriological and mycological characteristics of perennial waterholes in Old Oyo National Park (OONP), Nigeria. Coliform test was performed using the most probable number (MPN) technique and heterotrophic plate count (aerobic) by Pour Plate method while fungi count was done using the direct plate and dilution plate methods. Microbial populations were expressed as colony forming units per milliliter (cfu/ml). Different species of microflora were observed in the sampled waterholes includes Bacillus sp, Aspergillus spp, Actinobacter sp, Pseudomonas sp, Penicillium sp, Enterobacter sp, Klebsiella sp, Salmonella sp, Flavobacter sp, Seratia sp and Shigella sp. The presence of Escherichia coli observed in the study is an indication of recent faecal contamination. The mean values of microbial counts of the water samples were higher during the dry season with increased water concentration and more drinking from animals a probable cause. The presence of the various microflora observed in the study could pose a serious threat to both wildlife and public health. The study gave an insight to the health of the animals over seasons in the park and how it can affect health and population and a source of contaminants to all animals. There is need for continuous assessment of water quality in waterholes in OONP as a means of ensuring good ecological health.