The novelty of this study was to investigate for the first time in literature the influence of Bombyx mori silkworm cocoon’s age on the properties of silk fibroin-based materials, during all stages of cocoon processing to obtain the fibroin film. The study started with the premise that the cocoon age could cause modifications on fibroin properties during processing and, consequently, a possible interference on the characteristics of the final product. Characterizations were performed using batches of cocoons produced in different years, named C0 (fresh cocoons) and C6 (six-year-old cocoons). The influence of cocoon’s aging was observed on dialyzed dispersion regarding the molecular weight, particle size, and conformation. C6 films showed a more crystalline structure and higher thermal resistance than C0 films. The findings reported in this work are relevant for the reproducibility of fibroin-based materials, and the cocoon age is a key factor that should be considered in the preparation of fibroin-based materials.