Natural fiber-reinforced composites (NFRC) are becoming more popular as technological advance recognizes the need for eco-friendly materials. This work presents Giant ragweed (Ambrosia trifida) as a new composite reinforcement material into polylactic acid (PLA) matrix to form novel natural fiber-reinforced composites. Different types of retting techniques are utilized, and hydrogen peroxide retting is proven to provide the most optimized properties. The fibers are taken through subsequent extraction and grinding processes. Surface treatment of the ragweed fiber is conducted using maleic anhydride (MA), GLYMO, and AMEO silanes to decrease the fiber’s hydrophilic properties to have a better fiber/matrix interfacial adhesion. The bio-composite filaments are manufactured using single-screw and twin-screw extruders at 10wt.% fiber loading. The extrusion parameters like temperature and extruder speed were adjusted accordingly to obtain a fiber diameter of 1.65 mm. The test samples were 3D printed to ASTM standards D638 for tensile testing and D790 for flexural testing to study and compare mechanical characteristics using the Fused Deposition Modelling (FDM) technique. Morphological and thermal properties were characterized using scanning electron microscopy (SEM), Energy Dispersive X-ray Spectroscopy (EDAX), Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and thermomechanical analysis (TMA). The results demonstrate promising mechanical and thermal properties. Ultimately, this research provides the foundation for ragweed as a suitable reinforcement material by establishing an optimal processing framework for ragweed-PLA biocomposites.