As bacterial resistance continues to rise globally, the development of novel and sustainable antibacterial materials is becoming increasingly important. Azetidinium derivatives, known for their ability to disrupt bacterial membranes, were evaluated in this study for their antibacterial activity and potential application with nanocrystalline cellulose (CNC), a biodegradable, plant-based material that can act as a functional matrix. This work represents a preliminary screening phase of ongoing research, aimed at identifying the most promising azetidinium derivatives for future conjugation with CNC. Sulfate analysis by pH titration confirmed that conjugation and dialysis reduced or replaced sulfate groups on CNC, validating functionalization. The antibacterial performance of several azetidinium derivatives was evaluated against Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) using optical density (OD)-based growth inhibition assays. These assays were carried out with the Varioskan™ LUX Microplate Reader, which provided a high-throughput and reliable method for quantifying bacterial growth across multiple concentrations and replicates. Percentage inhibition values were calculated and analyzed through principal component analysis (PCA), hierarchical clustering, and heatmaps, offering both statistical and visual insights into activity trends. Compounds containing long hydrophobic chains and ester groups consistently demonstrated strong antibacterial activity, particularly against S. aureus, while CNC controls and simpler derivatives showed weaker effects. The heatmap revealed distinct inhibition patterns across concentrations, with S. aureus generally more sensitive and E. coli showing higher resistance. These patterns were consistent with PCA and clustering analyses, which grouped structurally similar compounds together and highlighted the role of hydrophobic chain length and ester functionalities in antibacterial performance.