The delivery of most therapeutic agents is rendered ineffective for the treatment of brain diseases due to the presence of the blood–brain barrier (BBB). The goal of this study was to investigate the effect of pre-infusion focused ultrasound (FUS) and microbubbles on the distribution of direct brain infusion in vivo. A single-element FUS transducer was used in all sonications, which were carried out immediately prior to direct infusion procedures. Mice received direct infusion of either Gadolinium-labeled albumin (Gd-albumin, 74 kDa) or adeno-associated virus (AAV, ∼4 MDa). The volumes of Gd-albumin at 30 min were deemed comparable (P = 0.334) between the direct infusion (DI)-only group and the FUS + DI group. At 120 min, the FUS + DI group showed significantly higher contrast-enhanced volume (9.76 ± 0.74 mm³) than the DI-only group (7.14 ± 0.34 mm³). For mice infused with AAV, the total volume of transduction was estimated as GFP-positive regions and FUS + DI group demonstrated significantly higher (P = 0.017) transduction efficiency in vivo. In conclusion, enhanced bio-distribution of directly infused agents was observed when the targeted region was pre-conditioned with FUS and microbubbles. Focused ultrasound has the potential, as an adjuvant technique, to significantly enhance direct brain infusion and achieve the desired therapeutic outcomes.