Antibiotics are among the most widely used pharmaceuticals, and their indiscriminate application has raised significant environmental and public health concerns. Rapid and cost-effective detection of antibiotic residues in surface water remains a major analytical challenge. However, limited studies have investigated the combined influence of multiple antibiotics on electrochemical signals during simultaneous detection. This study aims to evaluate the effects and interactions of azithromycin (AZI), tetracycline (TET), ampicillin (AMPIC), and penicillin (PEN) using factorial analysis while minimizing the number of required experiments. Electrochemical measurements were performed using square wave voltammetry (SWV) with a ZnO nanoparticle-modified carbon paste electrode under optimized conditions for AZI detection (potential range 0–1.5 V, amplitude 50 mV, step potential 5 mV, and frequency 15 Hz). The sensor exhibited a linear response in the range of 10-109 µM, with a limit of detection (LOD) of 3.22 µM and a limit of quantification (LOQ) of 9.77 µM. The factorial model showed strong agreement between predicted and experimental values and revealed that AMPIC significantly enhanced the AZI current signal, while interactions among antibiotics had moderate effects. In this work, factorial analysis was integrated with ZnO-modified electrochemical sensing to investigate the interactions of antibiotics with multiple analytes, and the findings demonstrate the potential of this approach for optimizing electrochemical detection methods in complex environmental matrices.