Here, we describe the synthesis and preliminary biological evaluation of novel N-unsubstituted and N-methylated 2-aryl benzimidazole derivatives that contain fluorinated or hydroxylated alkyl substituents in the 4-N-aryl position and different substitution patterns (H vs Br vs I) in the benzimidazole ring. For the selected compounds and for comparison purposes, the congener benzothiazoles were also tested. The cytotoxic effect of 11 benzazole derivatives was evaluated in a panel of human cancer cell lines, such as breast (MCF7), melanoma (A375), cervix (HeLa), and glioblastoma (U87). In general, the compounds exerted a moderate cytotoxic activity against all cells tested. In particular, for the A375 and HeLa cells, the N-unsubstituted benzimidazoles and displayed a better cytotoxic profile than the respective N-methylated benzimidazole congeners ( and ). The biodistribution of compound , which has shown the highest cytotoxic activity active in the U87 glioblastoma cells (IC50 = 45.2 ± 13.0), was evaluated in CD1 mice using its 18F-labeled counterpart (). These studies showed that compound can cross the blood brain barrier with a reasonable brain uptake (1.24 and 2.81%I.A./g at 5 and 60 min p.i., respectively), which is a crucial issue for systemic chemotherapy of glioblastoma. Altogether, the in vitro antitumoral activity of benzimidazole against the U87 cells and the ability of its 18F-congener to cross the blood brain barrier provide a strong rationale to consider the reported fluoroalkylated 2-aryl benzimidazoles as lead candidates for the generation of chemotherapeutic agents, in particular, against highly aggressive brain tumors such as glioblastoma.