Multifunctional and biofunctional coatings for medical devices are an attractive strategy towards tailoring the interactions of the device with the body, thereby influencing the host response, and the susceptibility to microbial colonization. Here we describe the development of a coating process to yield amphiphilic, lubricious coatings, resistant to bacterial colonization, based on chitosan. Chitosan-fatty acid derivatives were obtained by simultaneous O,N-acylation of chitosan with either linoleic, α-linolenic, or dilinoleic acid. Chemical characterization of new materials was carried out using 1H NMR, FTIR, and XPS. Surface properties of coated polyester samples were studied using SEM and contact angle measurements, which indicated that the incorporation of hydrophobic constituents into chitosan macromolecules led to a decrease of both surface roughness and water contact angle. Importantly, tribological testing demonstrated that these new coatings decrease the coefficient of friction due to the self-organization of fatty acid (from 0.53 for the neat chitosan to 0.35 for chitosan-fatty acid derivative). Meanwhile, preliminary bacterial colonization tests indicated significant—over 80%—reduction in E. coli colonization following coating with chitosan-linoleic and chitosan-α-linolenic derivatives. Finally, cytotoxicity and hemocompatibility studies confirmed that all amphiphilic chitosan-fatty acid derivatives were non-toxic and non-hemolytic. Collectively, our results demonstrate the potential of the developed coating strategy, particularly the chitosan-linoleic and chitosan-α-linolenic acid derivatives, for applications as biofunctional catheter coatings.