In this article, detailed procedure for the development of polylactic acid (PLA), hydroxyapatite (HAp) and chitosan (CS)-based biocompatible functional prototype has been outlined by using three-dimensional (3D) printing as a case study. The biocompatible composite-based feedstock filament (comprising of PLA-HAp-CS) has been prepared through twin-screw extruder (TSE) for open-source fused deposition modelling (FDM)-based 3D printer. This case study provides two-stage multifactor optimization: (a) for preparation of feedstock filament on TSE and (b) 3D printing on FDM based upon tensile and flexural samples. The results of study suggest that the best settings of input parameters for TSE are barrel temperature of 190°C, screw speed of 140 r/min and dead weight of 12 kg. Further, the optimized settings for FDM are layer thickness of 0.2 mm, deposition angle of 30/45° and infill density of 100%. The results have been supported by scanning electron microscopic analysis.