The performance of a biopolymer-amended subgrade soil during the service period still generates issues for pavement engineers, considering the deterioration under repeated traffic loading conditions leading to fatigue and rutting. The biopolymer stabilized soils exhibit brittleness after extended stabilization periods, and hence will be subjected to fatigue failure at higher loading cycles. In this study, chitosan (a crustacean biopolymer), extracted from shrimp shells is incorporated in clayey silt collected from the Kuttanad region of Kerala state, India. The chitosan–soil mix ratios adopted for the fatigue study are 1%, and 2% (by dry weight of the soil) and cured for 7, 14, and 28 days of curing periods. The untreated and chitosan-stabilized soil beam samples are subjected to flexural strength tests followed by repeated loading tests. In the fatigue testing machine, the beam samples are subjected to different load ratios of the modulus of rupture, and the loading cycles to failure are noted. The curing period and chitosan concentration positively influence the flexural strength and fatigue life of the amended samples. At the lowest stress ratio of 0.2, the fatigue life of 2% and 28-day cured samples are improved by 11.8 times compared to untreated soil. An important parameter affecting the fatigue life of stabilized layer(s) is the strain endured by the material. The strain value reduces by 22% for 2% samples at the highest curing period of 28 days for a stress ratio of 0.2. This research will facilitate a better understanding of biopolymer-treated subgrade soils by assisting pavement engineers in designing robust flexible pavements.