Phosphorylated chitosan, derived from the internal bone of Sepia kobiensis (Hoyle, 1885), has been investigated for its antimicrobial properties against oral pathogens. Chitosan's solubility in water and its biological effectiveness were enhanced through the alteration of its composition with orthophosphoric acid and dimethylformamide (DMF). Structural analysis for both chitosan and phosphorylated chitosan was performed using FTIR spectroscopy, field emission scanning electron microscopy (FESEM), and X-ray diffraction (XRD). The antimicrobial efficacy of chitosan and phosphorylated chitosan was assessed against clinically isolated human pathogens, including Streptococcus mutans, Escherichia coli, Pseudomonas aeruginosa, and Candida tropicalis. The highest inhibition zone, with a diameter of 23 ± 1.33 mm, was observed against P. aeruginosa for chitosan, while phosphorylated chitosan recorded a 25 ± 2.05 mm inhibition zone against E. coli. The findings of this study indicate that phosphorylated chitosan, with its potent antimicrobial properties, could serve as a possible antimicrobial drug for oral pathogens, sourced from a natural and sustainable origin.