Clostridium perfringens was analysed in soils from a tropical highland catchment and corresponding headwater streams at baseflow condition in order to understand the contribution of soils to the microbiological quality of stream water and the feasibility of using these streams as surrogate for negative control (reference streams). The concentrations of C. perfringens depended on the sample matrix. C. perfringens concentrations were significantly higher in the catchment soil than in the stream water (P < 0.05, n = 20). In addition, C. perfringens concentrations in the catchment soil remarkably predicted C. perfringens concentrations in the stream water (i.e., 82% of variations in C. perfringens concentrations in water were predicted by C. perfringens concentrations in soil; P < 0.05, n = 20). This suggests that the catchment soils contributed C. perfringens to the stream water. Despite the observed contamination, the concentrations of C. perfringens (geometric mean of 32 cfu/100 cm3) in the stream water was below the recommended safe level for tropical freshwater systems and extremely lower than that detected in anthropogenically influenced rivers. This concentration was defined as an acceptable level of disturbance, and a reference concentration that can serve as surrogate for negative control in the studied tropical environment.