The study of two new acylthiourea derivatives (Ar-CO-NH-CS-NH-R) are focused on the bonding interactions supported by X-ray, NMR, UV-Vis, Raman and IR spectroscopy, and NBO, AIM and Hirshfeld surface analysis. The RAHB model is proposed to explain some structural properties. The molecules are stabilized by intra-molecular NH center dot center dot center dot O bonds arranged in the lattice as centre-symmetric dimers held by inter-molecular NH center dot center dot center dot S bonds. The dimers are linked to each other through NH center dot center dot center dot O bonds giving rise to a chain, ribbon-like structure in the network. In vitro bacterial growth inhibition, biofilm formation, biosensor and biofilm metabolic activity were tested considering the broad bioactivity of acylthioureas.