Chitosan (CS), a natural material, shows excellent antimicrobial activity and is widely used in biomedical applications. The chitosan and Zirconium(Zr)-Hydroxyapatite (HAP) thin films were prepared with different concentrations of fish bone powder and tested for their antimicrobial activity. The bacterial organisms of Gram-positive (Staphylococcus aureus), Gram-negative (Escherichia coli), and fungal organisms (Saccharomyces cerevisiae) are used for antimicrobial tests. Antimicrobial susceptibility testing is performed to check the effectiveness of chitosan as drug and determine the sensitivity and resistance of pathogens towards it. Antimicrobial activity was assessed by viable cell count assay, which is a test performed to determine the number of actively dividing cells /growing cells in the sample. Results proved that the fish bone powder incorporated into thin films with the combination of CS-Zr-HAP had shown good inhibitory effect on microbial growth. The results showed that the synthesized chitosan-based Zirconium-Hydroxyapatite thin films inhibit the activity of Gram-negative bacteria, as Zirconium ions have a positive charge, which attacks the cell wall of E.coli, in turn, resulting in the organism’s death. A clear zone of inhibition is formed around the disc, which can be measured using a ruler; whereas Saccharomyces cerevisiae fungal organism doesn’t show growth inhibition, which indicates that the sample shows less effect or no effect on inhibition of some fungal species.