Combinatory therapy using two or more kinds of antibiotics is attracting considerable attention for inhibiting multi-drug resistant pathogenic bacteria. Although the therapy mostly leads to more powerful antimicrobial effects than using a single antibiotic (synergy), interference may arise from certain antibiotic combinations, resulting in the antimicrobial effect being suppressed (antagonism). Here, we present a microfluidic-based phenotypic screening chip to investigate combinatory antibiotic effects by automatically generating two orthogonal concentration gradients on a bacteria-trapping agarose gel. Computational simulations and fluorescence experiments together verify the simultaneous establishment of 121 concentration combinations, facilitating on-chip drug testing with stability and efficiency. Against Gram-negative bacteria, Pseudomonas aeruginosa, our chip allows the measurement of phenotypic growth levels, and enables various types of analyses for all antibiotic pairs to be conducted in 7 h. Furthermore, by providing a specific amount of susceptibility data, our chip enables the two reference models, Loewe additivity and Bliss independence, to be implemented, which classify the antibiotic interaction types into synergy or antagonism. These results suggest the efficacy of our chip as a cell-based drug screening platform for exploring the underlying pharmacological patterns of antibiotic interactions, with potential applications in guidance in clinical therapies and in screening other cell-type agents.