This study investigates the influence of deposition conditions in material extrusion (MEX) on fracture toughness, with a specific focus on the interlayer adhesion. A full factorial experimental design was employed, varying three key parameters: the deposition strategy, the extrusion multiplier, and the extruder speed. Fracture toughness was assessed using double cantilever beam tests, following ASTM D5528 standards. Additionally, the study explores the influence of load direction through various deposition strategies, including 0/90 and ± 45 orientations. To gain deeper insights, real-time thermal analysis was conducted during deposition, utilizing an infrared thermal camera. This allowed to investigate the effect of deposition conditions on temperature history. Subsequent examination of fracture surfaces post-testing was performed using optical and scanning electron microscopy. The findings reveal compelling evidence of the significant impact of the extrusion multiplier, printing speed, and deposition orientation on interlayer adhesion. In addition, the results indicated the presence of crystalline phase after deposition which was due to partially melting during depositions involving high material flow. This was due to the adoption of a semicrystalline filament. The adoption of the multidisciplinary approach enabled a better understanding of some phenomena occurring during the deposition (e.g., formation/existence of crystalline phase) that influence the adhesion behavior. These results underline the capability of such broad approach to analyze the influence of the processing conditions on the interlayer adhesion. Consequently, the developed analysis procedure represents a pivotal approach to study and optimize the MEX process and filament characteristics especially for semicrystalline polymers.