The presence of pathogenic free-living amoebae (FLA) such as Naegleria fowleri in freshwater environments is a potential public health risk. Although its occurrence in various water sources has been well reported, its presence and associated factors in biofilm remain unknown. In this study, the density of N. fowleri in biofilms spontaneously growing on glass slides fed by raw freshwater were followed at 32 °C and 42 °C for 45 days. The biofilms were collected with their substrata and characterized for their structure, numbered for their bacterial density, thermophilic free-living amoebae, and pathogenic N. fowleri. The cell density of N. fowleri within the biofilms was significantly affected both by the temperature and the nutrient level (bacteria/amoeba ratio). At 32 °C, the density remained constantly low (1–10 N. fowleri/cm2) indicating that the amoebae were in a survival state, whereas at 42 °C the density reached 30–900 N. fowleri/cm2 indicating an active growth phase. The nutrient level, as well, strongly affected the apparent specific growth rate (μ) of N. fowleri in the range of 0.03–0.23 h−1. At 42 °C a hyperbolic relationship was found between μ and the bacteria/amoeba ratio. A ratio of 106 to 107 bacteria/amoeba was needed to approach the apparent μmax value (0.23 h−1). Data analysis also showed that a threshold for the nutrient level of close to 104 bacteria/amoeba is needed to detect the growth of N. fowleri in freshwater biofilm. This study emphasizes the important role of the temperature and bacteria as prey to promote not only the growth of N. fowleri, but also its survival.