Environmental pollution by toxic heavy metals (HM) presents a real threat for aquatic medium and human health. Therefore aquatic ecosystem management requires early warning systems for on line monitoring. Microtechnologies can give rise to innovative bio-inspired hybrid microsensors, likely to meet this need and providing cost reductions by reducing reagents consumption and manufacturing cost. This work deals with a bacteria-based Love wave sensor, with enhanced properties provided by integration of a polydimethylsiloxane (PDMS) microfluidic network for a better control of the sample flow, and devoted to in situ monitoring of Cd(II) and Hg(II). Whole Escherichia coli (E. coli) bacteria are used as bioreceptor, mimicking in vivo enzymatic activity. They were immobilized on polyelectrolyte multilayer (PEM) films realized using layer by layer technique (LbL) with alternatively adsorption of positive and negative chains. The acoustic delay line was inserted into an electronic oscillation loop for real time monitoring. Compared to previous work, this paper deepens the results obtained with two types of microfluidic chips (measurements in static and dynamic modes), including analysis in terms of reproducibility. These results are analyzed and interpreted thoroughly leading to assumptions about the phenomena involved in the detection mechanisms. These hypotheses are validated through a pioneering study with atomic force microscopy (AFM), performed to characterize bacteria adhesion and to establish the relationship between bacteria morphological evolution and mechanical properties. AFM was chosen for its ability to maintain the bacteria alive during the experience without inducing irreversible damage. The resulting microsystem led to efficient HM detection, characterized by a reduced response-time (less than 60 s) and a detection limit inferior to 10−12 M. AFM measurements have demonstrated a high bacterial attachment and the stressing effect of toxic HM on bacterial morphological state. These results are consistent with those obtained from Love wave measurements.