Hydroxyapatite (HAP) is a biomaterial commonly used as a regenerative scaffold in bone, dental, and connective tissue engineering. Fish scales are a valuable source of hydroxyapatite due to their natural composition. In this study, fish scale waste was effectively converted into fish scale powder (FSP), which served as a viable source of hydroxyapatite. Subsequently, fish scale powder (FSP) combined with nano-hydroxyapatite (nHAP) was used to produce dental membranes as monolayers/bilayers. The physical and chemical characterization of the membranes was performed using various techniques, such as Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction analysis (XRD), field emission scanning electron microscopy (FESEM), energy dispersive X-ray spectroscopy (EDX), and mapping analysis. These membranes were loaded with methotrexate (MTX), and the MTX release after 30, 60, 120, and 240 minutes was evaluated by UV-Vis spectrophotometry. The data showed that the amount of MTX released increased over time, and the membranes exhibited high encapsulation efficiencies ranging from 81.47% to 96.43% at 240 minutes. These novel biocompatible and cost-effective membranes have significant potential for various applications in dentistry and related fields, improving the efficiency of the healing process by increasing the amount of drug released over time.