Strict distinguishability is the property of two systems to generate identical output signals only for the zero control input and the zero initial state vector. The objective of this paper is to characterize this property for single-output nonlinear control-affine systems. In this context, we first introduce the indistinguishability zone of the systems. This zone represents the set of control inputs and initial state vectors that make the output signals of the systems indistinguishable. The explicit identification of the elements of this set is proposed in the paper. From this result, necessary and sufficient conditions for strict distinguishability are then established under suitable assumptions.