Novel imidazoline benzimidazole derivatives containing diversely substituted phenoxy moieties were synthesized with the aim of evaluating their antitrypanosomal activity, DNA/RNA binding affinity and in vitro ADME properties. The presence of the diethylaminoethyl subunit in 18a–18c led to enhanced antitrypanosomal potency, particularly for 18a and 18c, which contain unsubstituted and methoxy-substituted phenoxy moieties. They were found to be > 2-fold more potent against African trypanosomes than nifurtimox. Fluorescence and CD spectroscopy, thermal denaturation assays and computational analysis indicated a preference of 18a–18c toward AT-rich DNA and their minor groove binding mode. Replacement of the amidine group with less basic and ionisable nitrogen-containing moieties failed to improve membrane permeability of the investigated compounds. Due to structural diversification, the compounds displayed a range of physico-chemical features resulting in variable in vitro ADME properties, leaving space for further optimization of the biological profiles. Display omitted •Novel imidazoline-substituted 2-arylbenzimidazoles were synthesized.•Diethylaminoethyl moiety in 18a–18c enhanced antitrypanosomal potency.•18a and 18c are > 2-fold more potent against T. brucei than nifurtimox.•18a–18c indicate preference toward AT-rich DNA and minor groove binding mode.•7a−7c, 17a−17c, and 18a–18c have favourable ADME properties.