The present document serves as the first evidence that describes the presence of amphizoic amoebae in cap, stalk and internal tissues such as gills of the edible Oyster mushrooms, Pleurotus sajor-caju, packaged, and sold for public consumption. Samples tested over a 9 month period from different vegetable markets of Lucknow city revealed that out of 180 mushrooms, 176 (97.77%) yielded amphizoic amoebae, namely, Schizopyrenus, Naegleria, Hartmannella, Acanthamoeba and Vanella sp. Four samples of internal tissues yielded 6 strains of amoebae. A total of 17 Naegleria strains were isolated, out of which 5 were identified as Naegleria fowleri and were tested for their pathogenicity. The strains 2BNf01 and 5BNf04 were mildly pathogenic and strains, 4ANf02i, 4ANf03 and 8ANf05i were highly pathogenic. It was also observed that the most frequently observed species of amoebae belonged to the genus Acanthamoeba and a total 103 Acanthamoeba strains recorded.