The protozoon Naegleria gruberi is able to carry out amoeboid locomotion at the water–air interface in a manner indistinguishable from that exhibited on solid substrata with the production of focal contacts and associated filopodia. The speed of locomotion at this interface can be modulated by changes in electrolyte concentrations; these speed changes are identical to those observed at a water-glass interface. The nature of the water–air interface is discussed leading to the hypothesis that surface tension alone could provide suitable properties for the adhesion and translocation of amoebae at this interface without necessitating specific, absorbed molecules. The temporary swimming flagellate stage of Naegleria is able to dock at the interface, make stable adhesions to it, and revert to the amoeboid phenotype. Conversely, amoebae resident at the water–air interface can transform to swimming flagellates and escape into the bulk liquid phase. We report the presence of Naegleria amoebae in the surface microlayers of natural ponds; thus, in freshwater bodies there may be active shuttling of Naegleria amoebae from the benthos to the surface microlayers by means of the non-feeding, swimming flagellate phenotype. The public health implication of this behaviour in the case of the pathogenic relative, Naegleria fowleri, is discussed.