Despite being biodegradable, paper packages have restricted use in food packaging because of their strong tendency to absorb moisture and their high permeability to liquids and gasses from the environment. Consequently, investigating the application of biodegradable biopolymers, such as nanofibrillated cellulose and chitosan, to enhance characteristics is a pertinent technique. This study developed paper from unbleached bamboo alkaline sulfite anthraquinone and methanol (ASAM) pulps by incorporating nanofibrillated cellulose (NFC) and the antimicrobial agent chitosan (CS) into the papermaking process, offering a sustainable solution for advanced food packaging systems. The objective of this research is to investigated the potential impact of varying concentrations of NFC (5% and 10%) and CS (0.5%, 1%, 1.5%, and 2%) on the physical, mechanical, thermal, barrier, and antimicrobial properties of unbleached bamboo ASAM pulp, considering 4.000 and 6.000 beating revolutions to enhance the mechanical, thermal, barrier, and antimicrobial properties. The reinforcement of NFC and CS has significant enhancements to the paper's properties. The results showed that incorporating 5% NFC and 1.5% chitosan at 6.000 beating revolutions has the optimum values of a tensile index and a burst index, where it reaches 85.16 Nm/g and 7.69 kPa m/g, respectively. Besides that, it exhibited sufficient thermal stability to be used for food packaging applications, with the onset temperature of thermal degradation about 258.28 °C. The smoothness and porosity showed increases of 11.93% and 96.35%, respectively. This reflects a decrease in air permeability. Additionally, the paper sheets demonstrated antimicrobial activity against various food-borne microorganisms a notable rise of 43.96% and 49.75% against Staphylococcus aureus and Candida albicans, respectively. It concluded that the ASAM-reinforced paper with these great properties exhibited a promising prospect in food packaging applications.