Microorganism assessment plays a key role in food quality and safety control but conventional techniques are costly and/or time consuming. Alternatively, electronic tongues (E-tongues) can fulfill this critical task. Thus, a potentiometric lab-made E-tongue (40 lipid sensor membranes) was used to differentiate four common food contamination bacteria, including two Gram positive (Enterococcus faecalis, Staphylococcus aureus) and two Gram negative (Escherichia coli, Pseudomonas aeruginosa). Principal component analysis and a linear discriminant analysis-simulated annealing algorithm (LDA-SA) showed that the potentiometric signal profiles acquired during the analysis of aqueous solutions containing known amounts of each studied bacteria allowed a satisfactory differentiation of the four bacterial strains. An E-tongue-LDA-SA model (12 non-redundant sensors) correctly classified 98 ± 5% of the samples (repeated K-fold-CV), the satisfactory performance of which can be attributed to the capability of the lipid membranes to establish electrostatic interactions/hydrogen bonds with hydroxyl, amine and/or carbonyl groups, which are comprised in the bacteria outer membranes. Furthermore, multiple linear regression models, based on selected subsets of E-tongue sensors (12–15 sensors), also allowed quantifying the bacteria contents in aqueous solutions (0.993 ± 0.011 ≤ R2 ≤ 0.998 ± 0.005, for repeated K-fold-CV). In conclusion, the E-tongue could be of great value as a preliminary food quality and safety diagnosis tool.