Chitosan is a well sought-after polysaccharide in biomedical applications due to its biocompatibility, biodegradability to non-toxic substances, and ease of fabrication into various configurations. However, alterations in the anti-bacterial properties of chitosan in various forms is not completely understood. The objective of this study was to evaluate the anti-bacterial properties of chitosan matrices in different configurations against two pathogens—Gram-positive Streptococcus mutans and Gram-negative Actinobacillus actinomycetemcomitans. Two-dimensional (2-D) membranes and three-dimensional (3-D) porous scaffolds were synthesized by air drying and controlled-rate freeze drying. Matrices were suspended in bacterial broths with or without lysozyme (enzyme that degrades chitosan). Influences of pore size, blending with Polycaprolactone (PCL, a synthetic polymer), and neutralization process on bacterial proliferation were studied. Transient changes in optical density of the broth, adhesion characteristics, viability, and contact-dependent bacterial activity were assessed. 3-D porous scaffolds were more effective in reducing the proliferation of S. mutans in suspension than 2-D membranes. However, no significant differences were observed on the proliferation of A. actinomycetemcomitans. Presence of lysozyme significantly increased the antibacterial activity of chitosan against A. actinomycetemcomitans. Pore size did not affect the proliferation kinetics of either species, with or without lysozyme. NaOH neutralization of chitosan increased bacterial adhesion whereas ethanol neutralization inhibited adhesion without lowering proliferation. Mat culture tests indicated that chitosan does not allow proliferation on its surface and it loses antibacterial activity upon blending with PCL. Results suggest that the chemical and structural characteristics of chitosan-based matrices can be manipulated to influence the interaction of different bacterial species.