This study aims to determine the influence polymeric branching in plasticized starch biopolymer (by varying the amylose/amylopectin ratio) and the influence of plasticiser on water activity of starch biopolymeric samples. Two types of starch were used; 80% amylose with 20% amylopectin (HA starch) and 20% amylose with 80% amylopectin (LA starch) with two different plasticisers, namely, glycerol and xylitol based on their number of hydroxyl (OH) groups (3 for glycerol and 5 for xylitol). The effect of branching was evident on the water activity after gelatinisation; HA starch was always lower in equilibrium moisture content than the LA starch – which is significantly affected by relative humidity. This could be due to the overall increase in the amorphous nature of amylopectin which provides greater free volume for water to be retain and thus increasing the equilibrium water content. Also, at lower water activities, all samples with lower plasticizer content tend to have greater moisture content and this behaviour was opposite at higher plasticiser content. This ‘inversion’ behaviour is explained via the strong linkages that both glycerol and xylitol molecules can form with amylose chains at low amounts, causing a reduction in free volume and loss of bound water from the polymer matrix. From a molecular point of view, this translates into the well-known anti-plasticisation and it appears that there exists a ‘threshold’ water activity where the interaction between the starch and plasticiser is partially replaced by a stronger starch and water interaction causing greater equilibrium water in the system.