Laguna de Bay or Laguna Lake is one of the six major lakes in the Philippines to be in close contact with population activities due to the expansion of urban settlements in the immediate cities surrounding the lake, thus pushing the population to settle upon its shores. To date, there are no data showing the biodiversity of free-living amoebae (FLA) present in this lake. The present study aims to isolate and identify the FLA present in Laguna de Bay, Philippines. Thirty subsurface water samples were taken from Laguna De Bay using random purposive sampling in May 2018 and were examined for amoebic growth under light microscopy (LM). Results show that 8 out of 30 (26.6%) water samples were positive for amoebic growth and were further tested for more advanced data and genetic variation of the species. Initial molecular analysis using polymerase chain reaction (PCR) and sequencing showed the presence of potentially pathogenic FLA Naegleria australiensis (MK418954). The detection of potential pathogenic FLA in lakes and dams may prove useful in preventing and controlling possible human infections in the country. More data from this study will aid in public awareness and establishing safety guidelines and control programs.