The early stages of fouling development on artificial substrates were examined for spatial-temporal variation in the commercial and touristic harbours (use destinations) of the port of Livorno (Tuscany, Italy). The experiment was carried out by submerging two types of experimental ropes with different surface textures, considering three times of submersion. Particular attention was paid to the colonization dynamics of non-indigenous species (NIS). The type of rope did not significantly affect fouling development. However, when the NIS assemblage and the whole community were taken into account, the colonization of ropes varied depending on the use destination. The touristic harbour exhibited a degree of fouling colonization higher than the commercial one. NIS were observed in both harbours since the beginning of colonization, eventually achieving higher population densities in the touristic harbour. The use of experimental ropes represents a promising quick cost-effective tool for monitoring of NIS presence in port environments.