Phenotypic diversity in bacterial flagella-induced motility leads to complex collective swimming patterns, appearing as traveling bands with transient locally enhanced cell densities. Traveling bands are known to be a bacterial chemotactic response to self-generated nutrient gradients during growth in resource-limited microenvironments. In this work, we studied different parameters of Escherichia coli (E. coli) collective migration, in particular the quantity of bacteria introduced initially in a microfluidic chip (inoculum size) and their exposure to antibiotics (ampicillin, ciprofloxacin, and gentamicin). We developed a hybrid polymer-glass chip with an intermediate optical adhesive layer featuring the microfluidic channel, enabling high-content imaging of the migration dynamics in a single bacterial layer, i.e., bacteria are confined in a quasi-2D space that is fully observable with a high-magnification microscope objective. On-chip bacterial motility and traveling band analysis was performed based on individual bacterial trajectories by means of custom-developed algorithms. Quantifications of swimming speed, tumble bias and effective diffusion properties allowed the assessment of phenotypic heterogeneity, resulting in variations in transient cell density distributions and swimming performance. We found that incubation of isogeneic E. coli with different inoculum sizes eventually generated different swimming phenotype distributions. Interestingly, incubation with antimicrobials promoted bacterial chemotaxis in specific cases, despite growth inhibition. Moreover, E. coli filamentation in the presence of antibiotics was assessed, and the impact on motility was evaluated. We propose that the observation of traveling bands can be explored as an alternative for fast antimicrobial susceptibility testing.