Daptomycin (DAP) is an antimicrobial drug of the cyclic lipopeptide class. It has broad activity against a wide range of gram-positive bacterial species. Here, the electrochemical behavior of daptomycin was investigated for the first time using a disposable pencil graphite electrode (PGE) in a 0.5 M H2SO4 supporting electrolyte solution. The interaction with DNA was examined using three different techniques: electrochemical, spectrometric, and molecular docking. The surface characterization of PGE was conducted using techniques such as electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and scanning electron microscopy (SEM). In this study, a sensitive, selective, accurate, and green electroanalytical method for the quantification of DAP was developed. DAP gave an irreversible anodic signal of about + 1.02 V on the PGE surface against an Ag/AgCl reference electrode with the cyclic voltammetry technique. The developed method gave a linear response in the concentration range of 0.166 mg/L to 1.328 mg/L and the limit of detection (LOD) and limit of quantification (LOQ) values were 0.032 mg/L, and 0.107 mg/L. The interaction of DAP with DNA was studied using voltammetric, spectrophotometric, and molecular docking techniques. The evaluation of the results obtained with all three techniques revealed that the interaction of the DAP molecule with DNA is mediated by groove binding. This study offers an innovative approach to antimicrobial discovery by thoroughly investigating the electrochemical behavior of DAP and its interaction with DNA using three different techniques, highlighting its unique contribution to the field.