Summary: Microbial mats are highly structured and diverse communities, and one of the earliest‐known life assemblages. Mat bacteria interact within an environment marked by strong geochemical gradients and fluctuations. We examined natural mat systems for the presence of autoinducers involved in quorum sensing, a form of cell–cell communication. Our results revealed that a diverse array of N ‐acylhomoserine lactones (AHLs) including C ₄ ‐ to C ₁₄ ‐AHLs, were identified from mat extracts using mass spectrometry (MS), with further confirmation by MS/MS‐collision‐induced dissociation (CID), and additions of external standards. Microelectrode measurements showed that mats exhibited diel pH fluctuations, ranging from alkaline (pH 9.4) during daytime (net photosynthesis) to acidic (pH 6.8) during darkness (net respiration/fermentation). Under laboratory conditions, AHLs having shorter acyl‐chains were degraded within the time frame that daily alkaline pH (> 8.2) conditions exist in mats. Intensive sampling of mats after full day‐ or night‐time incubations revealed that accumulations of extractable shorter‐chain AHLs (e.g. C ₈ ‐ and C ₁₀ ‐AHLs) were significantly ( P < 0.001) diminished during daytime. Our study offers evidence that stabilities of AHLs under natural conditions may be influenced by the proximal extracellular environment. We further propose that the ancient periodicity of photosynthesis/respiration in mats may potentially drive a mechanism for diel differences in activities of certain autoinducers, and hence bacterial activities mediated through quorum sensing.