Introduction Better understanding the effect of temperature is key in the framework of the current global warming of earth. Temperature has indeed a strong influence on growth rate, which exhibits a non-monotonic behaviour with a temperature at which the growth rate is maximum. Even if the optimal growth temperature varies between microbial types (psychrophiles, mesophiles, thermophiles) this non-monotonic behaviour holds true for virtually any microbial culture known to date. In this contribution, we question if such a generic property could be sustained by a fundamental physical principle, namely the non-equilibrium fluctuation relations. Methods To challenge theory development, we grew Escherichia coli K12 NCM3722 strain (∆motA), in minimal medium (Modified MOPS culture with 6 amino acids) containing 500 mg/L of glucose as the sole energy source in 48-wells microplate with experiments with continuous optical density measurements performed at increasing temperature from 28 to 44 °C with 1°C steps. Growth rates were computed from at least 25 replicated cultures at each temperature. Correlation between optical density measurements, dry mass and microbial concentrations (measured by flow cytometry) were carefully determined in parallel Erlenmeyer experiments to allow the derivation of microbial yields and associated thermodynamic balances. Results and discussion Building on a previous contribution [1], we have extended the Microbial Transition State (MTS) [2]; to include temperature dependency based on an explicit theoretical ground. Then, we questioned the predictions of the model using our experimental measurements and associated thermodynamic computations. According to the extended MTS theory (eMTS) proposed in this contribution, the non-monotonic behaviour of growth rate according to temperature is explained by the trade-off between two types of antagonistic effects: the dissipation of free energy, acting as a driving force, and the thermal stability of microbial structures. Importantly, the model correctly represents experimental growth data at each temperature not only for the exponential growth phase but also of the decay phases, which constitutes a unique feature to our knowledge. Dissipation and activation energies associated to the growth of E.coli culture are determined and discussed. This contribution therefore supports and shows how the non-equilibrium fluctuation relation governs the temperature behaviour of microbial cultures.