Bacteria responds to changing chemical and thermal environment by moving towards or away from a particular location. In this report, we looked into thermal gradient generation and response of E. coli DH5α cells to thermal gradient in the presence and in the absence of spherical gold nanoparticles (size: 15 to 22 nm) in a static microfluidic environment using a polydimethylsiloxane (PDMS) made microfluidic device. A PDMS-agarose based microfluidic device for generating thermal gradient has been developed and the thermal gradient generation in the device has been validated with the numerical simulation. Our studies revealed that the presence of gold nanoparticles, AuNPs (0.649 μg/mL) has no effect on the thermal gradient generation. The E. coli DH5α cells have been treated with AuNPs of two different concentrations (0.649 μg/mL and 0.008 μg/mL). The thermotaxis behavior of cells in the presence of AuNPs has been studied and compared to the thermotaxis of E.coli DH5α cells in the absence of AuNPs. In case of thermotaxis, in the absence of the AuNPs, the E. coli DH5α cells showed better thermotaxis towards lower temperature range, whereas in the presence of AuNPs (0.649 μg/mL and 0.008 μg/mL) thermotaxis of the E. coli DH5α cells has been inhibited. The results show that the spherical AuNPs intervenes in the themotaxis of E. coli DH5α cells and inhibits the cell migration. The reason for the failure in thermotaxis response mechanism may be due to decreased F-type ATP synthase activity and collapse of membrane potential by AuNPs, which, in turn, leads to decreased ATP levels. This has been hypothesized since both thermotaxis and chemotaxis follows the same response mechanism for migration in which ATP plays critical role. © 2016, Springer Science+Business Media New York.