Understanding the dynamic environmental microniches of biofilms will permit us to detect, manage and exploit these communities. The components and architecture of biofilms have been interrogated in depth; however, little is known about the environmental microniches present. This is primarily because of the absence of tools with the required measurement sensitivity and resolution to detect these changes. We describe the application of ratiometric fluorescent pH-sensitive nanosensors, as a tool, to observe physiological pH changes in biofilms in real time. Nanosensors comprised two pH-sensitive fluorophores covalently encapsulated with a reference pH-insensitive fluorophore in an inert polyacrylamide nanoparticle matrix. The nanosensors were used to analyse the real-time three-dimensional pH variation for two model biofilm formers: (i) opportunistic pathogen Pseudomonas aeruginosa and (ii) oral pathogen Streptococcus mutans. The detection of sugar metabolism in real time by nanosensors provides a potential application to identify therapeutic solutions to improve oral health.