This study aimed to understand the structure-property relationship in chitosan (CHI) based layer-by-layer (LbL) films and to correlate the surface properties with the biological characteristics of the multilayers. LbL films of CHI were prepared using two different types of polyacids, i.e. a polyphenol, tannic acid (TA) and a polycarboxylic acid, poly(acrylic acid) (PAA). The chemical nature and topology of the polyacid was found to be critical on LbL growth, stability under physiologically related conditions, surface morphology, wettability and drug release properties of the multilayers. The differences in surface properties provided remarkably different biological properties such as anti-adhesiveness against protein adsorption and antibacterial activity for Ciprofloxacin (CIP) incorporated films against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Listeria monocytogenes (L. monocytogenes). Linearly growing TA/CHI films with relatively smooth surfaces resisted more against protein adsorption when the topmost layer and the protein carried like charges. On the other hand, exponentially growing PAA/CHI multilayers did not exhibit antiadhesive behaviour. Thick and loose PAA/CHI multilayers were able to release higher amount of CIP, thus presented enhanced antibacterial activity compared to thin and more intense TA/CHI films. Finally, we showed that pH affected the extent of association among the layers as well as drug release and antibacterial properties of the films. Our findings showed that TA/CHI films are more promising as antiadhesive surfaces, whereas PAA/CHI multilayers are more suitable for preparation of antibacterial surfaces. The fundamental findings of this study may form a basis for preparation of thin films for drug delivery applications and modification of biomaterial surfaces.