Legionella is a ubiquitous water environmental organism. Isolation/detection of Legionellae has been reported worldwide. However, there is no reported isolation of Legionellae in countries in Africa with the exception of South Africa. The aim of this study was to survey the surface, ground waters and air conditioner water systems (ACWs) in Osogbo, Nigeria for the presence of Legionella pneumophila by cultural isolation method. A total of 313 water samples from the streams (surface water), wells (groundwater) and ACWS were cultured on buffered charcoal yeast extract (BCYE) medium. The isolates were identified by Gram staining techniques, biochemical tests and demonstration of pore forming activity. Polymerase chain reaction for amplification of 0.52 kb fragment of the part of dot/icm region of L. pneumophila was used for the final identification. The results obtained showed that the prevalence of L. pneumophila from streams (8%) was statistically significant when compared to wells (1%) (Chi square, P < 0.05). L. pneumophila was not isolated from any of the water samples taken from air-conditioner water systems. The study concludes that L. pneumophila is present in natural aquatic environment in Nigeria.